U.S. patent number 6,573,295 [Application Number 09/990,637] was granted by the patent office on 2003-06-03 for bicyclic signal transduction inhibitors, compositions containing them & uses thereof.
This patent grant is currently assigned to Ariad Pharmaceuticals, Inc.. Invention is credited to Regine Bohacek, Charles Joseph Eyermann, Tomi K. Sawyer, William C. Shakespeare, Rajeswari Sundaramoorthi, Michael G. Yang.
United States Patent |
6,573,295 |
Shakespeare , et
al. |
June 3, 2003 |
Bicyclic signal transduction inhibitors, compositions containing
them & uses thereof
Abstract
This invention concerns compounds for inhibiting intracellular
signal transduction, especially intracellular signal transduction
mediated by one or more molecular interactions involving a
phosphotyrosine-containing protein. This invention also relates to
pharmaceutical compositions containing the compounds and
prophylactic and therapeutic methods involving pharmaceutical and
veterinary administration of the compounds. The compounds are of
the formula ##STR1## as defined herein.
Inventors: |
Shakespeare; William C.
(Framingham, MA), Yang; Michael G. (Chestnut Hill, MA),
Sundaramoorthi; Rajeswari (Watertown, MA), Bohacek;
Regine (Boston, MA), Eyermann; Charles Joseph (Sudbury,
MA), Sawyer; Tomi K. (Southborough, MA) |
Assignee: |
Ariad Pharmaceuticals, Inc.
(Cambridge, MA)
|
Family
ID: |
22320348 |
Appl.
No.: |
09/990,637 |
Filed: |
November 21, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
438601 |
Nov 12, 1999 |
|
|
|
|
Current U.S.
Class: |
514/450; 514/456;
514/470; 549/355; 549/404; 549/467 |
Current CPC
Class: |
A61P
37/08 (20180101); C07F 9/4021 (20130101); C07D
313/08 (20130101); C07F 9/12 (20130101); C07F
9/3873 (20130101); A61P 19/10 (20180101); A61P
9/08 (20180101); A61P 29/00 (20180101); A61P
35/00 (20180101); C07F 9/3882 (20130101); C07F
9/65527 (20130101); A61P 37/00 (20180101); A61P
43/00 (20180101); C07C 237/48 (20130101); A61P
9/00 (20180101); C07F 9/3834 (20130101); C07C
2602/12 (20170501); C07C 2601/14 (20170501) |
Current International
Class: |
C07C
237/00 (20060101); C07C 237/48 (20060101); C07F
9/12 (20060101); C07F 9/38 (20060101); C07F
9/655 (20060101); C07F 9/40 (20060101); C07F
9/00 (20060101); C07D 313/08 (20060101); C07D
313/00 (20060101); A61K 031/335 (); A61K 031/35 ();
A61K 031/34 (); C07D 311/04 (); C07D 307/78 () |
Field of
Search: |
;514/456,470,450
;549/404,467,355 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
5464863 |
November 1995 |
Nagamine et al. |
5929107 |
July 1999 |
Natsugari et al. |
6211203 |
April 2001 |
Amschler |
6302837 |
October 2001 |
De Nanteuil et al. |
6316494 |
November 2001 |
Jacobsen et al. |
6323238 |
November 2001 |
Yoo et al. |
6333349 |
December 2001 |
Brendel et al. |
6346527 |
February 2002 |
Takenaka et al. |
6395738 |
May 2002 |
Ohshima et al. |
6399657 |
June 2002 |
Braunlich et al. |
|
Foreign Patent Documents
|
|
|
|
|
|
|
WO 96/30332 |
|
Oct 1996 |
|
WO |
|
WO 97/12903 |
|
Apr 1997 |
|
WO |
|
WO 97/31016 |
|
Aug 1997 |
|
WO |
|
WO 99/24442 |
|
May 1999 |
|
WO |
|
WO 99/47529 |
|
Sep 1999 |
|
WO |
|
Other References
Wang et al., "Design of a high affinity peptidomimetic opiod
agonist from peptide pharmacophore models." ---(1998) Bioorganic
& Medicinal Chemistry Letters, 8(19): 2685-2688. .
Tamami et al., "Pyrazine-based polymeric complex of
oxodiperoxochromium (VI) compound as a new stable, mild, efficient
and versatile oxidant in organic synthesis." ---(1997) Tetrahedron,
NL Elsevier Science, 53 (23): 7889-7896. .
Reiner Luckenback, "Beilsteins Handbuch der organischen Chemie."
---(1987) Springer-Verlag, 4th Edition, 5th Suppl., vol. XVIII,
Part 9, p. 650, paragraph 1. .
Reiner Luckenback, "Beilsteins Handbuch der organischen Chemie."
---(1985) Springer-Verlag, 4th Edition, 4th Suppl., vol. XVIII,
Part 4, p. 2819, paragraph 5. .
Bernard Prager et al., "Beilsteins Handbuch der organischen
Chemie." ---(1929) Springer-Verlag, 4th Edition, vol. XII, p. 1207,
paragraph 3..
|
Primary Examiner: Lambkin; Deborah C.
Attorney, Agent or Firm: Berstein; David L.
Parent Case Text
RELATED APPLICATIONS
This application is a continuation application of U.S. Ser. No.
09/438,601 filed Nov. 12, 1999, ABN which is a continuation in part
of U.S. Ser. No. 60/108,106 filed Nov. 12, 1998, the full contents
of which are incorporated herein by reference.
Claims
What is claimed is:
1. A compound of the formula: ##STR43##
wherein Y is ##STR44## each occurrence of G is --O--, --S-- or
-NR-; R.sup.6 comprises -APO.sub.3 RR, --OPO.sub.3 RR, -ASO.sub.3
R, --OSO.sub.3 R, -ACO.sub.2 R, -A-tetrazole, -A--N--(PO.sub.3
RR)(PO.sub.3 RR), -ASO.sub.2 NRR, -ACOCF.sub.3, --(C.dbd.O)J,
--C(R)(J)(K) or --C(Z)(J)(K); where each occurrence of A is
independently a covalent bond, -G-M- or -(M).sub.m -; each
occurrence of M is an independently selected, substituted or
unsubstituted, methylene moiety, and any M--M moiety may be
electronically saturated or unsaturated; each n is independently
0,1, 2, 3, 4 or 5; each m is independently 0, 1 or 2; J and K are
independently -APO.sub.3 RR, --OPO.sub.3 RR, -ASO.sub.3 R,
--OSO.sub.3 R, -ACO.sub.2 R, -A-tetrazole, -ASO.sub.2 NRR,
-(M).sub.n --NRR or -(M).sub.n --OR; Z is a halogen; R.sup.7 and
R.sup.8 are independently R, --CN, --NO.sub.2, Z, J, -A(M).sub.n
aliphatic, -G(M).sub.n aliphatic , -(M).sub.n COCF.sub.3,
-(M).sub.n OH, -(M).sub.n COOR, -A-(M).sub.n NRR, -G(M).sub.q NRR,
-(M).sub.n CHO, -A(M).sub.n N(R)(CO)R, -A(M).sub.n N(R)(CO)GR,
-G(M).sub.q N(R)(CO)R, -G-(M).sub.q N(R)(CO)GR, -A-(M).sub.n
--CO-NRR, or -G(M).sub.n -CO-NRR, where the aliphatic groups may be
substituted or unsubstituted; or R.sup.7 is a covalent bond to an
R.sup.4 substituent of X to form an aliphatic, aryl or heterocyclic
ring of 4 to 8 atoms; each occurrence of R (unnumbered) represents
hydrogen or an aliphatic, heteroaliphatic, aryl, heteroaryl,
(aryl)aliphatic-, or (heteroaryl)aliphatic-moiety, each of which
(other than hydrogen) may be substituted or unsubstituted; q is an
integer from 1 to 8; X is: --(CR.sup.3 R.sup.4).sub.m -- or
--NR.sup.4 --; R.sup.3 hydrogen, R(CO)NR--, RRN(CO)NR--, RSO.sub.2
NR--, RCSNR--, RRNCSNR--, RRNSO.sub.2 NR--, ROCONR--, RRN--, or
##STR45##
and, R.sup.4 is hydrogen, aliphatic, cycloaliphatic-(M).sub.n -,
aryl-(M).sub.n -, heterocyclic-(M).sub.n -, R--SO.sub.2 M.sub.n -,
(RO--CO)(M).sub.n - or (RRN--CO)(M).sub.n -, where the aliphatic,
cycloaliphatic, aryl or hete4ocyclic moiety is substituted or
unsubstituted; p is 1, 2, 3 or 4; R.sup.9, R.sup.10, R.sup.11 and
R.sup.12 are independently -(M).sub.n Z, -(M).sub.n R, -(M).sub.n
GR, -(M).sub.n WR, -(M).sub.n WGR, or -(M).sub.n W--COR, or R.sup.9
and R.sup.10 are covalently linked together to form an aliphatic,
heterocyclic or aryl fused ring; and, U and W are independently
--CO--, --CS--, -M-, --SO--, or --SO.sub.2 --; or a
pharmaceutically acceptable derivative thereof.
2. A compound of claim 1 of the formula: ##STR46##
3. A compound of claim 1 wherein each n is independently 0, 1 or
2.
4. A compound of claim 1 wherein X is --CH(NH.sub.2)--.
5. A compound of claim 1 of the formula ##STR47##
where R.sup.5 comprises a substituted or unsubstituted lower
aliphatic or alkoxyl or is a substituted or unsubstituted
-(M).sub.n -aryl or -(M).sub.n -heterocyclic group.
6. A compound of claim 5 wherein R.sup.5 comprises -(M).sub.n
CH.sub.3, -(M).sub.n aryl, -(M).sub.n heterocyclic, -(M).sub.n CN,
-(M).sub.n COOR, --O--(M).sub.n CH.sub.3, --O(M).sub.n aryl,
--O(M).sub.n heterocyclic, --O(M).sub.n CN, or --O(M).sub.n COOR,
where n is 0, 1, 2, 3, 4, or 5.
7. A compound of claim 5 wherein R.sup.5 is a substituted or
unsubstituted methyl, ethyl, i-propyl, n-propyl, n-butyl, isobutyl,
t-butyl, cyclobutyl, n-amyl, sec-amyl, isoamyl, cyclopentyl,
n-hexyl, sec-hexyl, isohexyl, cyclohexyl or benzyl.
8. A compound of claim 5 wherein R.sup.5 comprises
--(CH.sub.2).sub.n CH.sub.3, --(CH.sub.2)(CH.sub.2).sub.n aryl,
--(CH.sub.2)(CH.sub.2).sub.n heterocyclic,
--(CH.sub.2)(CH.sub.2).sub.n CN, --(CH.sub.2)(CH.sub.2).sub.n COOR,
--O(CH.sub.2).sub.n CH.sub.3, --O(CH.sub.2)(CH.sub.2).sub.n aryl,
--O(CH.sub.2)(CH.sub.2).sub.n heterocyclic,
--O(CH.sub.2)(CH.sub.2).sub.n CN, or --O(CH.sub.2)(CH.sub.2).sub.n
COOR, where n is 0, 1, 2, 3, 4, or 5.
9. A compound of claim 8 wherein R.sup.5 comprises --CH.sub.2 CN,
--CH.sub.2 phenyl, --CH.sub.2 aryl, --CH.sub.2 heterocyclic,
--CH.sub.2 COOR, --CH.sub.2 COOR, --(CH.sub.2).sub.3 COOR,
--(CH.sub.2).sub.4 COOR, where R is H, lower alkyl or benzyl.
10. A compound of claim 1 of the formula ##STR48##
wherein R.sup.5 comprises --O--(M).sub.n CH.sub.3, --O(M).sub.n
aryl, --O(M).sub.n heterocyclic, --O(M).sub.n CN, or --O(M).sub.n
COOR, where n is 0, 1, 2, 3, 4, or 5.
11. A compound of claim 10 wherein R.sup.5 comprises
--O(CH.sub.2).sub.n CH.sub.3, --O(CH.sub.2)(CH.sub.2).sub.n aryl,
--O(CH.sub.2)(CH.sub.2).sub.n heterocyclic,
--O(CH.sub.2)(CH.sub.2).sub.n CN, or --O(CH.sub.2)(CH.sub.2).sub.n
COOR, where n is 0, 1, 2, 3, 4, or 5.
12. A compound of claim 10 wherein R.sup.5 comprises
--O-(substituted or unsubstituted lower alkyl or benzyl).
13. A compound of claim 1 of the formula ##STR49##
where R.sup.4 is hydrogen, substituted or unsubstituted aliphatic
(which may be branched, unbranched or cyclic), substituted or
unsubstituted aryl-(M).sub.n -, substituted or unsubstituted
heterocyclic-(M).sub.n -, or (CO.sub.2 R)(M).sub.n -.
14. A compound of claim 13 wherein R.sup.4 is -(M).sub.n (CO)OR,
-(M).sub.n SO.sub.2 R, -(M).sub.n (CO)NRR, or -(M).sub.n
(tetrazole).
15. A compound of claim 14 wherein R.sup.4 is --CH.sub.2 COOR,
--CH.sub.2 SO.sub.2 R, --CH.sub.2 (CO)NRR, or -tetrazole.
16. A compound of claim 14 wherein each R is independently H, lower
alkyl or benzyl.
17. A compound of claim 1 of the formula ##STR50##
where R.sup.4 is hydrogen, substituted or unsubstituted aliphatic
(which may be branched, unbranched or cyclic), substituted or
unsubstituted aryl-(M).sub.n -, substituted or unsubstituted
heterocyclic-(M).sub.n -, or (CO.sub.2 R)(M).sub.n -.
18. A compound of claim 17 wherein R.sup.4 is hydrogen.
19. A compound of claim 1 of the formula ##STR51##
20. A compound of any of claims 5-12 wherein each R is H.
21. A compound of any of claims 1-19 wherein M is --CH.sub.2
--.
22. A compound of any of claims 1-19, wherein Y comprises
##STR52##
23. A compound of claim 22 wherein R.sup.6 comprises --OR,
-APO.sub.3 RR, --OPO.sub.3 RR, -ASO.sub.3 R, --OSO.sub.3 R,
-ACO.sub.2 R, -A-tetrazole, -ASO.sub.2 NRR, -ACOCF.sub.3,
--C(R)(J)(K) or --C(Z)(J)(K); and R.sup.7 and R.sup.8 are
independently H, --CN, --NO.sub.2, halogen, J, -A-(M).sub.n
aliphatic, -G-(M).sub.n aliphatic, -(M).sub.n COCF.sub.3,
-(M).sub.n OH, -(M).sub.n COOR, -A-(M).sub.n NRR, -G-(M).sub.q NRR,
-(M).sub.n CHO, -A-(M).sub.n N(R)(CO)R, -G-(M).sub.q N(R)(CO)R,
-A-(M).sub.n -CO-NRR, or -G-(M).sub.n -CO-NRR, where the aliphatic
groups may be substituted or unsubstituted; or R.sup.7 is a
covalent bond to an R.sup.4 substituent of X to form a ring of 4 to
8 atoms.
24. A compound of claim 22, wherein R.sup.6 comprises --OR,
-APO.sub.3 RR, --OPO.sub.3 RR, -ACO.sub.2 R, -ACOCF.sub.3 or
--C(R)(J)(K); A comprises --CH.sub.2 --, --OCH.sub.2 --, --CF.sub.2
--, --CHF--, --CHOH-- or a covalent bond; each R H, or substituted
or unsubstituted lower alkyl or substituted or unsubstituted
benzyl; and, R.sup.7 and R.sup.8 are independently H, J,
-A-(M).sub.n substituted or unsubstituted aliphatic, -G-(M).sub.n
substituted or unsubstituted aliphatic, -(M).sub.n COCF.sub.3,
-(M).sub.n OH, -(M).sub.n COOR, -A-(M).sub.n NRR, -(M).sub.n CHO,
-A-(M).sub.n N(R)(CO)R or -A-(M).sub.n --CO--NRR.
25. A compound of claim 22 wherein R.sup.6 comprises --OH,
--PO.sub.3 RR, --OPO.sub.3 RR, --CH.sub.2 PO.sub.3 RR, CF.sub.2
PO.sub.3 RR, --OCH.sub.2 CO.sub.2 R, --NHCH.sub.2 CO.sub.2 R,
--CH.sub.2 CO.sub.2 R, --CF.sub.2 CO.sub.2 R, --CH.sub.2 SO.sub.3
R, CF.sub.2 SO.sub.3 R, --CH.sub.2 COCF.sub.3, --CF.sub.2
COCF.sub.3, --CH(PO.sub.3 RR).sub.2, --CH(OH)(PO.sub.3 RR),
--CH(NH.sub.2)(PO.sub.3 RR), --CH(CO.sub.2 R).sub.2, --CF(CO.sub.2
R).sub.2, --CH(PO.sub.3 RR)(CO.sub.2 R"), --CH(PO.sub.3
RR)(SO.sub.3 R"), --CH(PO.sub.3 RR)(SO.sub.2 NH.sub.2),
--CH(SO.sub.2 NH.sub.2).sub.2, or --CH(SO.sub.3 RR).sub.2.
26. A compound of claim 25 in which one or more R group in the
--PO.sub.3 RR, --OPO.sub.3 RR, --CH.sub.2 PO.sub.3 RR, CF.sub.2
PO.sub.3 RR, --OCH.sub.2 CO.sub.2 R, --NHCH.sub.2 CO.sub.2 R,
--CH.sub.2 CO.sub.2 R, --CF.sub.2 CO.sub.2 R, --CH.sub.2 SO.sub.3
R, --CF.sub.2 SO.sub.3 R, --CH.sub.2 COCF.sub.3, --CF.sub.2
COCF.sub.3, --CH(PO.sub.3 RR).sub.2, --CH(OH)(PO.sub.3 RR),
--CH(NH.sub.2)(PO.sub.3 RR), --CH(CO.sub.2 R).sub.2, --CF(CO.sub.2
R).sub.2, --CH(PO.sub.3 RR)(CO.sub.2 R), --CH(PO.sub.3 RR)(SO.sub.3
R), --CH(PO.sub.3 RR)(SO.sub.2 NH.sub.2), --CH(SO.sub.2
NH.sub.2).sub.2, or --CH(SO.sub.3 RR).sub.2 moiety is H.
27. A compound of claim 25 in which one or more R in the --PO.sub.3
RR, --OPO.sub.3 RR, --CH.sub.2 PO.sub.3 RR, --CF.sub.2 PO.sub.3 RR,
--OCH.sub.2 CO.sub.2 R, --NHCH.sub.2 CO.sub.2 R, --CH.sub.2
CO.sub.2 R, --CF.sub.2 CO.sub.2 R, --CH.sub.2 SO.sub.3 R,
--CF.sub.2 SO.sub.3 R, --CH.sub.2 COCF.sub.3, --CF.sub.2
COCF.sub.3, --CH(PO.sub.3 RR).sub.2, --CH(OH)(PO.sub.3 RR),
--CH(NH.sub.2)(PO.sub.3 RR), --CH(CO.sub.2 R).sub.2, --CH(PO.sub.3
RR)(CO.sub.2 R), --CH(PO.sub.3 RR)(SO.sub.3 R), --CH(PO.sub.3
RR)(SO.sub.2 NH.sub.2), --CH(SO.sub.2 NH.sub.2).sub.2, or
--CH(SO.sub.3 RR).sub.2 moiety is -(M).sub.m CH.sub.2 Z, -(M).sub.m
CHZ.sub.2, -(M).sub.m CZ.sub.3, --R.sup.15, -M--O--CO--OR.sup.15 or
-M--O--CO--OR.sup.15, where Z is halogen and R.sup.15 is
substituted or unsubstituted lower aliphatic, aryl or
heterocyclic.
28. A compound of claim 27 in which R.sup.15 is methyl, ethyl,
n-propyl, -propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl,
benzyl or substituted benzyl.
29. A compound of claim 25 wherein R.sup.7 and R.sup.8 are H.
30. A compound of claim 25 wherein R.sup.7 is J, -A-(M).sub.n
(substituted o aliphatic, aryl or heterocyclic), -G-(M).sub.n
(substituted or unsubstituted aliphatic, aryl or heterocyclic),
-(M).sub.n COCF.sub.3, -(M).sub.n OH, -(M).sub.n COOR, -A-(M).sub.n
NRR, -(M).sub.n CHO, -A-(M).sub.n N(R)(CO)R, -A-(M).sub.n --NRR or
-A-(M).sub.n --CO--NRR; and R.sup.8 is H.
31. A compound of claim 25 wherein R.sup.7 is lower alkyl, lower
alkenyl, --OH, --NH.sub.2, --NO.sub.2, --CN, --NHR, --NHCOR, --CHO,
--CH.sub.2 CHO, --PO.sub.3 RR, --OPO.sub.3 RR, --CH.sub.2 PO.sub.3
RR, --CF.sub.2 PO.sub.3 RR, --OCH.sub.2 CO.sub.2 R, --NHCH.sub.2
CO.sub.2 R, --CH.sub.2 CO.sub.2 R, --CF.sub.2 CO.sub.2 R,
--SO.sub.3 R, --CH.sub.2 SO.sub.3 R, --CF.sub.2 SO.sub.3 R,
--COCF.sub.3, --COCH.sub.2 F, --COCF.sub.2 H, --CF.sub.2 COCF.sub.3
or --SO.sub.2 NH.sub.2.
32. A compound of claim 31 in which at least one R group in
--PO.sub.3 RR, --OPO.sub.3 RR, --CH.sub.2 PO.sub.3 RR, --CF.sub.2
PO.sub.3 RR, --OCH.sub.2 CO.sub.2 R, --NHCH.sub.2 CO.sub.2 R,
--CH.sub.2 CO.sub.2 R, --CF.sub.2 CO.sub.2 R, --SO.sub.3 R,
--CH.sub.2 SO.sub.3 R, or --CF.sub.2 SO.sub.3 R is H.
33. A compound of claim 31 in which at least one R group in
--PO.sub.3 RR, --OPO.sub.3 RR, --CH.sub.2 PO.sub.3 RR, --CF.sub.2
PO.sub.3 RR, --OCH.sub.2 CO.sub.2 R, --NHCH.sub.2 CO.sub.2 R,
--CH.sub.2 CO.sub.2 R, --CF.sub.2 CO.sub.2 R, --SO.sub.3 R,
--CH.sub.2 SO.sub.3 R, or --CF.sub.2 SO.sub.3 R is -(M).sub.m
--CH.sub.2 Z, -(M).sub.m --CHZ.sub.2, -(M).sub.m --CZ3, --R.sup.15,
-M--O--CO--R.sup.15 or -M--O--CO--OR.sup.15, where Z is halogen and
R.sup.15 is substituted or unsubstituted lower aliphatic, aryl or
heterocyclic.
34. A compound of claim 33 which R.sup.15 is methyl, ethyl,
n-propyl, -propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl,
benzyl or substituted benzyl.
35. A compound of claim 29 wherein R.sup.6 comprises -APO.sub.3 RR
or --OPO.sub.3 RR and R.sup.7 is -A-(M).sub.n -aliphatic or
-G-(M).sub.n -aliphatic, where the aliphatic moiety is substituted
or unsubstituted.
36. A compound of claim 35 wherein R.sup.6 comprises --OPO.sub.3
H.sub.2.
37. A compound of claim 22 wherein R.sup.6 and R.sup.7 are
independently selected from J and K.
38. A compound of claim 22 wherein R.sup.6 is --C(R)(J)(K).
39. A compound of claim 38 wherein R is H.
40. A compound of claim 38 wherein J is --PO.sub.3 RR.
41. A compound of claim 40 in which one or both R groups is H.
42. A compound of claim 40 in which one or both R groups is
R.sup.15, -(M).sub.m --CH.sub.2 Z, -(M).sub.m --CHZ.sub.2,
-(M).sub.m --CZ.sub.3, -M--O--CO--R.sup.15 or -M--O--CO--OR.sup.15,
where Z is halogen and R.sup.15 is substituted or unsubstituted
lower aliphatic, aryl or heterocyclic.
43. A compound of claim 42 in which R.sup.15 is methyl, ethyl,
n-propyl, -propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl,
benzyl or substituted benzyl.
44. A compound of claim 22, wherein each of R.sup.9, R.sup.10,
R.sup.11 and R.sup.12 is independently -(M).sub.n Z, -(M).sub.n R,
-G(M).sub.n R, -(M).sub.n WR or -(M).sub.n W-GR.
45. A compound of claim 44 wherein one or more of the R groups
contain at least one substituent selected from halo, hydroxy, or a
substituted or unsubstituted aliphatic, amino, amido or sulfonamido
moiety.
46. A compound of claim 44 wherein one or more of R.sup.9,
R.sup.10, R.sup.11, and R.sup.12 is a substituted aliphatic moiety
containing at least one substituent selected from substituted or
unsubstituted cycloaliphatic, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, --CO.sub.2 R, --CO--NRR,
and --OR.
47. A compound of claim 44, wherein one or more of R.sup.9,
R.sup.10, R.sup.11, and R.sup.12 comprises -(M).sub.n
(cycloaliphatic), -(M).sub.n (substituted or unsubstituted aryl),
-(M).sub.n (substituted or unsubstituted heteroaryl), -(M).sub.n
CHO, -(M).sub.n CONH.sub.2, -(M).sub.n CSNH.sub.2, -(M).sub.n
SONH2, -(M).sub.2 SO.sub.2 NRR, -(M).sub.n OR, -(M).sub.n (lower
aliphatic), -(M).sub.n --C(OR)RR, or -(M).sub.n --C.dbd.CRR.
48. A compound of claim 44, wherein one or more of R.sup.9,
R.sup.10, R.sup.11, and R.sup.12 comprises -G(M).sub.n (aliphatic),
-G(M).sub.n (cycloaliphatic), -G(M).sub.n (substituted or
unsubstituted aryl), -G(M).sub.n (substituted or unsubstituted
heteroaryl), -G(M).sub.n CHO, -G(M).sub.n CONH.sub.2, -G(M).sub.n
CSNH.sub.2, -G(M).sub.n SONH.sub.2, -G(M).sub.n SO.sub.2 NRR,
-G(M).sub.n OR, -G(M).sub.n (lower aliphatic), -G(M).sub.n
--C(OR)RR, or -G(M).sub.n --C.dbd.CRR.
49. A compound of claim 48 wherein -G(M).sub.n comprises
--OCH.sub.2 --, --SCH.sub.2 -- or --NRCH.sub.2 --.
50. A compound of claim 44, wherein one or more of R.sup.9,
R.sup.10, R.sup.11, and R.sup.12 comprises methyl,
--(CH.sub.2).sub.q R.sup.13 where q is 1-7 and R.sup.13 comprises
methyl; i-propyl; i-butyl; t-butyl; cycloaliphatic; phenyl;
substituted phenyl; naphthyl; substituted naphthyl; a 5, 6 or
7-membered heterocyclic ring or a bicyclic heterocylic moiety.
51. A compound of claim 44, wherein one or more of R.sup.9,
R.sup.10, R.sup.11, and R.sup.12 comprises -(M).sub.n W--NH--R and
one or more of R.sup.9, R.sup.10, R.sup.11, and R.sup.12 comprises
--O(M).sub.m (aliphatic).
52. A compound of claim 44 of the formula: ##STR53##
53. A compound of claim 44, wherein one or more of R.sup.9,
R.sup.10, R.sup.11, and R.sup.12 comprises -(M).sub.n
(CO)--NH--R.
54. A compound of claim 44, wherein one or more of R.sup.9,
R.sup.10, R.sup.11, and R.sup.12 comprises --CONH--R.
55. A compound of claim 44, wherein one or more of R.sup.9,
R.sup.10, R.sup.11, and R.sup.12 comprises --(CH.sub.2).sub.m
CONH--R.
56. A compound of claim 1 of the formula: ##STR54##
where R.sup.5 comprises a substituted or unsubstituted lower
aliphatic moiety, and R.sup.9 comprises -(M).sub.n W--NH--R and
R.sup.10 comprises --O(M).sub.m (aliphatic).
57. A compound of claim 56, wherein R.sup.9 comprises --CONH--R and
R.sup.10 comprises --OM-cycloaliphatic or --OM-branched chain
aliphatic.
58. A compound of claim 56, wherein R.sup.9 comprises --CH.sub.2
CONH--R and R.sup.10 comprises --OM-cycloaliphatic or --OM-branched
chain aliphatic.
59. A compound of claim 44, wherein Y comprises: ##STR55## R.sup.6
comprises -APO.sub.3 RR, -ASO.sub.3 R, -ACO.sub.2 R, -ASO.sub.2
NRR, -ACOCF.sub.3, or --C(R)(J)(K); and, R.sup.7 is H, --CN,
--NO.sub.2, halogen, J, -A-(M).sub.n substituted or unsubstituted
aliphatic, -(M).sub.n COCF.sub.3, -(M).sub.n OH, -(M).sub.n COOR,
-A-(M).sub.n NRR, -(M).sub.n CHO, -A-(M).sub.n N(R)(CO)R or
-A-(M).sub.n --CO--NRR.
60. A compound of claim 59, wherein R.sup.6 comprises
--OPO(OH).sub.2, --PO(OH).sub.2, --OCH.sub.2 COOH, --CF.sub.2
PO(OH).sub.2, or --CH(PO.sub.3 H.sub.2).
61. A compound of claim 44, wherein R.sup.7 comprises H.
62. A compound of claim 44, wherein R.sup.7 comprises CHO.
63. A compound of claim 44, wherein R.sup.7 comprises J.
64. A compound of claim 44, wherein R.sup.7 comprises -A-(M).sub.n
substituted or unsubstituted aliphatic.
65. A compound of claim 44, wherein R.sup.6 comprises
--OPO(OH).sub.2 and R.sup.7 is H.
66. A compound of claim 64, wherein (M).sub.n is
(CH.sub.2).sub.n.
67. A compound of claim 66, wherein n is 1.
68. A compound of claim 59, wherein one or more (unnumbered) R
groups comprise -(M).sub.m --CH.sub.2 Z, --(M).sub.m CHZ.sub.2,
-(M).sub.m CZ.sub.3, -M--O--CO--R.sup.15 or -M--O--CO--OR.sup.15,
where Z is halogen and R.sup.15 is a substituted or unsubstituted
lower aliphatic, aryl or heterocyclic group.
69. A compound of any of claims 1-19, which binds to a given SH2
domain with a value of less than about 50 .mu.M.
70. A compound of claim 69, which binds to a given SH2 domain with
a IC.sub.50 value of less than, about 20 .mu.M.
71. A compound of claim 69 wherein the SH2 domain is from a Src,
Fyn, Lck, Yes, Blk, Lyn, Fgr, Hck, Yrk, ZAP-70, Syk, STAT or Abl
protein.
72. A composition comprising a compound of any of claims 1-19, or a
pharmaceutically acceptable derivative thereof, and a
pharmaceutically acceptable excipient.
73. A method for inhibiting SH2-mediated signal transduction in a
mammal in need thereof which comprises administering to the mammal
a pharmaceutical composition of claim 72.
74. The method of claim 73, wherein the pharmaceutical composition
contains a compound which specifically binds to an SH2 domain of
Src, ZAP-70, Syk, or STAT 6.
75. The method of claim 73, wherein said SH2-mediated signal
transduction is mediated by a PDGF receptor protein, EGF receptor
protein, HER2/Neu receptor protein, fibroblast growth factor
receptor protein, focal adhesion kinase protein, p130 protein, or
p68 protein.
76. The method of claim 73, wherein the mammal has a proliferative
disease, restenosis, osteoporosis, inflammation, allergies, or
cardiovascular disease.
77. The method of claim 73, wherein the mammal has a cancer.
78. A method of treating a patient who has a proliferative disease,
restenosis, osteoporosis, inflammation, allergic reaction, or
cardiovascular disease, the method comprising administering to the
patient a therapeutically effective amount of a composition of
claim 72.
79. A method of treating a patient who has a cancer, the method
comprising administering to the patient a therapeutically effective
amount of a composition of claim 72.
80. A method for inducing immunosuppression in a patient the method
comprising administering to the patient an amount of a composition
of claim 72 sufficient to cause immunosuppression.
81. A compound of the structure: ##STR56## wherein each of R.sup.9
and R.sup.10 is independently halo, R, --OR, --SR, --NRR, --COR, or
-(M).sub.n W--NHR; each occurrence of M is an independently
selected, substituted or unsubstituted, methylene moiety; each n is
independently 0, 1, 2, 3, 4 or 5; each occurrence of R (unnumbered)
represents hydrogen or an aliphatic, heteroaliphatic, aryl,
heteroaryl, (aryl)aliphatic-, or (heteroaryl)aliphatic-moiety, each
of which (other than hydrogen) may be substituted or unsubstituted;
and, W is --CO--, --CS--, -M-, --SO--, or --SO.sub.2 --.
Description
FIELD OF THE INVENTION
This invention concerns a new class of compounds which have a broad
range of useful biological and pharmacological activities. In
particular, these compounds are useful for inhibiting intracellular
signal transduction, especially intracellular signal transduction
mediated by one or more molecular interactions involving a
phosphotyrosine-containing protein. This invention also relates to
pharmaceutical compositions containing the compounds and
prophylactic and therapeutic methods involving pharmaceutical and
veterinary administration of the compounds.
BACKGROUND OF THE INVENTION
Cellular signal transduction, i.e., the series of events leading
from extracellular events to intracellular sequelae, is an aspect
of cellular function in both normal and disease states. Numerous
proteins that function as signal transducing molecules have been
identified, including receptor and non-receptor tyrosine kinases,
phosphatases and other molecules with enzymatic or regulatory
activities. These molecules generally demonstrate the capacity to
associate specifically with other proteins to form a signaling
complex that can alter cell activity.
Signaling proteins often contain domain(s) of conserved sequence
which constitute catalytic domains such as kinase or phosphatase
domains, or serve as non-catalytic modules that direct
protein:protein or other inter- or intramolecular interactions
during signal transduction. Such domains include among others, Src
homology 2 ("SH2") and phosphotyrosine interaction ("PI") domains.
SH2 and PI domains recognize, i.e., bind to, proteins containing
characteristic peptide sequences which include one or more
phosphorylated tyrosine ("pTyr") residues. Significant information
related to such domains, proteins containing them, the production
of proteins containing such domains (including protein fragments
and fusion proteins), the characteristic peptide sequences which
they recognize and the biological and/or clinical role played by
the interactions of such proteins has been described in the
scientific literature. See e.g. U.S. Pat. No. 5,667,980,
PCT/US97/02635 ("Cell-Based Assay") and WO 97/39326 ("In Vitro
Fluorescence Polarization Assay") and references cited therein for
additional background information on SH2 and PI domains, inhibition
of intermolecular interactions mediated by such domains, assays and
related topics.
The protein domains of the tyrosine kinase, Src, gave rise to the
"Src homology" ("SH") nomenclature and illustrate this class of
proteins. At least nine members of the Src family of tyrosine
kinases have been identified to date in vertebrates including Src
(alternatively known as c-src and pp6oc-src), Fyn, Yes, Lyn, Hck,
Fgr, Blk and Yrk.
Sequence analysis of the Src tyrosine kinases reveals that each
family member contains an N-terminal membrane anchor, a poorly
conserved "unique" region of 40-70 amino acids, a Src homology 3
(SH3) domain of about sixty amino acids capable of protein--protein
interactions with proline-rich sequences and a Src homology 2 (SH2)
domain comprising about 100 amino acid residues which mediates
binding of the Src family member of phosphotyrosine-(pTyr)
containing peptides and proteins (reviewed in Superti-Furga, FEBS
Lett. 369:62-66 (1995). Several cognate phosphoproteins known to
bind the Src SH2 domain include middle T antigen, PDGF receptor,
EGF receptor, and focal adhesion kinase (FAK). See Courtneidge et
al, J. Virol. 65:3301-3308 (1991); Moi et al. EMBO J. 12:2257-2264
(1993); Luttrell et al. Proc. Natl. Acad. Sci.
USA 91:83-87 (1994); and Xing et al, Mol. Biol. Cell 5:413421
(1994). For additional information on other SH2 domains (including,
e.g., ZAP-70, Syk, Shc, Tsk, Btk, VAV, Grb2, Crk, STATs) and PI
domain-containing proteins, see WO 97/39326 and references cited
therein.
The molecular structure of several SH2 domains has been solved and,
in particular, the molecular structure of certain SH2 domains in
complex with a phosphotyrosine-containing peptide or peptide analog
has been elucidated. See Waksman et al, Cell 72:779-790 (1993); Xu
et al. Biochemistry 34:2107-2121 (1995); Hatada et al, Nature
377(6544), 32-38 (1995). Whereas the general consensus sequence of
Src family SH2-binding peptides, for example, comprises a
pTyr-X--X--(Leu/Ile) motif, SH2 domain binding specificity is
thought to be influenced significantly by the specific amino acids
located carboxy-terminal to the pTyr residue. For example, the
pp6oc-src, Fyn, Lck and Fgr SH2 domains prefer the sequence
pTyr-Glu-Glu-Ile. See Songyang et al, Cell 72:767-778 (1993).
Crystallographic data concerning pp60c-src SH2 in complex with
synthetic peptides has revealed, in particular, two important
binding determinants for binding to phosphotyrosine-containing
proteins or peptides: the phosphotyrosine binding site which is
electropositive in nature such that phosphotyrosine binding is
stabilized and the lipophilic binding site which stabilizes binding
of pTyr+3 residues within the phosphotyrosine-containing peptides
via hydrophobic contacts. Reviewed by Brown and Cooper, Biochim.
Biophys. Acta 1287 (2-3):121-149 (1996).
Structural studies of phosphotyrosine-containing peptides complexed
with isolated SH2 domains has provided information regarding the
molecular interactions of peptide ligands with the SH2 domain
peptidyl binding site. Recent attempts have been made to
extrapolate these data to design novel peptide ligands and
peptidomimetic agonists of SH2-mediated signaling. For example,
Plummer et al reported that incorporation of C-terminal D-amino
acid residues to tripeptide SH2 domain ligands increases affinity
relative to their L-amino acid-containing counterparts. See Plummer
et al, Drug Design Discovery 13:7581 (1996). Burke et al reported
that hexapeptides containing
difluoro-(phosphoromethyl)phenylalanine bound SH2 domains with high
relative affinity compared to analogous pTyr peptides and were
resistant to naturally-occurring cellular phosphatases. Studies of
the pTyr residue of peptide agonists of the Src SH2 domain have
shown that that phosphate ester is important for molecular
recognition, and that significant loss in binding occurs when it is
replaced with sulfate, carboxylate, nitro, hydroxy or amino groups.
See Gilmer et al, J Biol Chem 269:31711-31719 (1994).
Many signaling pathways which play critical roles in disease
processes are mediated by the binding of a
phosphotyrosine-containing protein or protein domain with an SH2 or
other protein receptor for a tyrosine-phosphorylated domain.
Pharmaceutical agents which interfere with signaling mediated by
such molecules, e.g., which interfere with the formation or
stability of such signaling complexes, may be used for precise
intervention in these complex biological processes in order to
treat or prevent the diseases or pathological effects mediated by
such signaling. Such interference may be achieved through a variety
of mechanisms, including competitive inhibition of a
phosphotyrosine-containing ligand with its receptor (e.g., with an
SH2-containing protein), inhibition of phosphorylation of the
tyrosine residue of such a ligand, inhibition of activation of a
kinase which catalyzes the phosphorylation of a ligand in a
signaling pathway, etc.
Compounds that can enter cells and block a signal transduction
pathway of interest, such as an SH2-mediated pathway, would be of
great interest as reagents for biological research and for
pharmaceutical and veterinary uses.
SUMMARY OF THE INVENTION
This invention concerns compounds of Formula I, or pharmaceutically
acceptable derivatives thereof: ##STR2##
in which
Y is ##STR3##
G is --O--, --S-- or --NR--;
R.sup.6 comprises --OR, -APO.sub.3 RR', --OPO.sub.3 RR', -ASO.sub.3
R, --OSO.sub.3 R, -ACO.sub.2 R, -A-tetrazole, -A--N--(PO.sub.3
RR')(PO.sub.3 RR')', -ASO.sub.2 NRR', -ACOCF.sub.3, --(C.dbd.O)J,
--C(R)(J)(K) or --C(Z)(J)(K);
where each occurrence of A is independently a covalent bond, -G-M-
or -(M).sub.m -;
each occurrence of M is an independently selected, substituted or
unsubstituted, methylene moiety, and any M-M' moiety may be
electronically saturated or unsaturated and/or may be part of a
3-8-membered ring. Illustrative "M" moieties include --CH.sub.2 --,
--CHF--, --CF.sub.2 --, --CHOH--, --CH(Me)--, etc.
Each n is independently 0, 1, 2, 3, 4 or 5 (in many embodiments n
is 0, 1 or 2);
each m is independently 0, 1 or 2;
J and K are independently selected from the group consisting of
-APO.sub.3 RR', --OPO.sub.3 RR, -ASO.sub.3 R, --OSO.sub.3 R,
-ACO.sub.2 R, -A-tetrazole, -ASO.sub.2 NRR', -(M).sub.n NRR' and
-(M).sub.n OR;
Z is a halogen (i.e., F, Cl, Br or I);
R.sup.7 and R.sup.8 are independently R, --CN, --NO.sub.2, Z, J,
-A(M).sub.n aliphatic, -G(M).sub.n aliphatic, -(M).sub.n
COCF.sub.3, -(M).sub.n OH, -(M).sub.n COOR, -A-(M).sub.n NRR,
-(M).sub.q NRR, -(M).sub.n CHO, -A(M).sub.n N(R)(CO)R', -A(M).sub.n
N(R)(CO)GR', -G(M).sub.q N(R)(CO)R', -G-(M).sub.q N(R)(CO)G'R',
-A-(M).sub.n --CO--NRR', or -GM).sub.n --CO--NRR', where the
aliphatic groups may be substituted or unsubstituted; or R.sup.7 is
a covalent bond to an R.sup.4 substituent of X forming an
aliphatic, aryl or heterocyclic ring of 4 to 8 atoms (including,
for example a 5-membered nitrogen-containing ring of an indole
moiety).
Each occurrence of R (unnumbered) represents hydrogen or an
aliphatic, heteroaliphatic, aryl, heteroaryl, (aryl)aliphatic-, or
(heteroaryl)aliphatic-moiety, each of which (other than hydrogen)
may be substituted or unsubstituted, e.g., with any of the various
substituents listed, illustrated or otherwise disclosed herein.
While each occurrence of "R" within a given compound is thus
independently selected, where multiple R groups are depicted in the
same figure or moiety, the various R groups are generally marked R,
R', R" and so on, as a reminder that they may be the same or
different. (The same is true in the case of numbered "R" groups and
other variables such as "m", "n", "M", etc. where apostrophes are
used for the same purpose. Note also that the n M groups in a
"M.sub.n " moiety may be the same or different from one
another.)
q is an integer from 1 to 8, and in many embodiments is 1, 2 or
3;
X is: --(CR.sup.3 R.sup.4).sub.m - or --NR.sup.4 --;
R.sup.3 is hydrogen, R(CO)NR'--, RR N(CO)NR"--, R'SO.sub.2 NR"--,
R'CSNR--, RR'NCSNR"--, RR'NSO.sub.2 NR"--, R'OCONR--, RR'N--, or
##STR4##
R.sup.4 is hydrogen, aliphatic (which may be branched, unbranched
or cyclic), cycloaliphatic-(M).sub.n -, aryl-(M).sub.n -,
heterocyclic(M).sub.n -, RSO.sub.2 (M.sub.n)-, (CO.sub.2
R)(M).sub.n - or (RR'N--CO)(M).sub.n, where the aliphatic,
cycloaliphatic, aryl and heterocyclic groups are substituted or
unsubstituted;
B is ##STR5##
where
E is M, G or one of the following: ##STR6##
p is 1, 2, 3 or 4;
R.sup.9, R.sup.10, R.sup.11 and R.sup.12 are independently
-(M).sub.n Z, -(M).sub.n R, -(M).sub.n GR, -(M).sub.n WR or
-(M).sub.n WGR, including, among others, moieties such as R, --OR,
--SR, CHO, --OR,--COOH (or amide, ester, carbamate, urea, oxime or
carbonate thereof), --NH.sub.2 (or substituted amine, amide, urea,
carbamate or guanidino derivative therof, halo, trihaloalkyl,
cyano, --SO.sub.2 --CF.sub.3, --OS(O).sub.2 F, --OS(O).sub.2 R,
--SO.sub.2 --NHR, --NHSO.sub.2 R, sulfate, sulfonate, aryl and
heteroaryl moieties. Alternatively, R.sup.10 and R.sup.11 are
covalently linked together to form an aliphatic, hetercyclic or
aryl fused ring, typically of 5-7 members. For example, in some
embodiments, R.sup.10 and R.sup.11 comprise -G-(M).sub.n -G'-, as
illustrated by the following structure for B where, for the sake of
example, each M is --CH.sub.2 -- and n is 3: ##STR7##
where in some cases G is and G' is --S--, for example.
R.sup.14 is R (H is generally preferred); and,
U and W are independently --CO--, --CS--, -M-, --SO--, or
--SO.sub.2 --:
or a pharmaceutically acceptable derivative thereof.
Compounds of Formula I thus include compounds having the following
structures: ##STR8##
and comprise a number of subgenera of particular interest.
Representative subgenera are illustrated in the examples which
follow.
One subgenus includes compounds in which at least one R.sup.4
moiety is H and at least one R.sup.3 moiety is either H or
NH.sub.2. Compounds of the latter sort include those in which X is
##STR9##
Also of interest are the subgenera of compounds in which the
nitrogen atom of the moiety X is further elaborated, as depicted
below: ##STR10##
where R.sup.5 comprises a substituted or unsubstituted, lower
(i.e., containing 1-8 carbon atoms) aliphatic or alkoxyl group, or
is a substituted or unsubstituted -(M).sub.n -aryl or -(M).sub.n
-heterocyclic (including e.g., substituted and unsubstituted phenyl
or benzyl group, or a homolog and heterocyclic analog thereof,
including e.g., 2-naphthyl, 3-indolyl, and 1-imidazolyl).
Such compounds are further illustrated by the subset thereof in
which R.sup.5 comprises -(M).sub.n CH.sub.3, -(M).sub.n aryl,
(M).sub.n heterocyclic, (M).sub.n CN, (M).sub.n COOR, where n is 0,
1, 2, 3, 4, or 5. For instance, in some such compounds R.sup.5 is a
substituted or unsubstituted methyl, ethyl, n-propyl, i-propyl,
n-butyl, sec-butyl, t-butyl, n-pentyl, sec-pentyl, i-pentyl, cyclo
pentyl, etc. or benzyl moiety. In other such compounds R.sup.5
comprises --(CH.sub.2).sub.n CH.sub.3, --(CH.sub.2)(CH.sub.2).sub.n
aryl, --(CH.sub.2)(CH.sub.2).sub.n heterocyclic,
--CH.sub.2)(CH.sub.2).sub.n CN or --(CH.sub.2)(CH.sub.2).sub.n
COOR, where n again is 0, 1, 2, 3, 4, or 5. Examples of such
compounds include those in which R.sup.5 comprises GH.sub.2 CN,
dCH.sub.2)CO.sub.2 R, --(CH.sub.2).sub.2 CO.sub.2 R,
--(CH.sub.2).sub.3 CO.sub.2 R, --(CH.sub.2).sub.4 CO.sub.2 R, where
R is H, lower alkyl or benzyl.
In some embodiments of compounds of the structure ##STR11##
R.sup.5 comprises --O(M).sub.n CH.sub.3, --O(M).sub.n aryl,
--O(M).sub.n heterocyclic, --O(M).sub.n CN, or --O(M).sub.n COOR,
where n is 0, 1, 2, 3, 4, or 5. In specific cases, R.sup.5
comprises --O(CH.sub.2).sub.n CH.sub.3,
--(CH.sub.2)(CH.sub.2).sub.n aryl, --O(CH.sub.2)(CH.sub.2).sub.n
heterocyclic, --O(CH.sub.2)(CH.sub.2).sub.n CN, or
--O(CH.sub.2)(CH.sub.2).sub.n COOR. In numerous cases, R.sup.5
comprises --O-(substituted or unsubstituted lower alkyl or
benzyl.
Another subgenus of interest includes amides of the formula:
##STR12##
where R.sup.4 is hydrogen, substituted or unsubstituted aliphatic
(which may be branched, unbranched or cyclic), substituted or
unsubstituted aryl-(M).sub.n -, substituted or unsubstituted
heterocyclic-(M).sub.n -, or (CO.sub.2 R)(M).sub.n -. Such
compounds are illustrated by those in which R.sup.4 is -(M).sub.n
(CO)OR, -(M).sub.n SO.sub.2 R, -(M).sub.n (CO)NRR', or -(M).sub.n
(tetrazole), including, for example, compounds in which R.sup.4 is
--CH.sub.2 COOR, --CH.sub.2 SO.sub.2 R, --CH.sub.2 (CO)NRR, or
-tetrazole. Simple members of this subgenus are those in which the
R group(s) of R.sup.4 is (are independently) H, lower alkyl (e.g.,
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tbutyl,
etc.) or benzyl.
Another subgenus includes ureas of the formula: ##STR13##
where R.sup.1, R.sup.2, R.sup.4, R .sup.14, Y and m are defined as
above. Thus, R.sup.4 may be simply H or may be a more complex
R.sup.4 moiety such as are noted above.
Another subgenus includes amides of the formula: ##STR14##
In many examples of all of the foregoing compounds, one or more R
moieties (R', R" etc) are H. Also, in many compounds of interest,
R.sup.14 is H.
Compounds of Formula I, including, among others, compounds of the
various subgenera described above, include those in which Y
comprises ##STR15##
Such compounds in which R.sup.6 comprises --OR, -APO.sub.3 RR',
--OPO.sub.3 RR', --ASO.sub.3 R, --OSO.sub.3 R, -ACO.sub.2 R,
-A-tetrazole, -A--N-(PO.sub.3 RR')(PO.sub.3 RR)', --ASO.sub.2 NRR',
-ACOCF.sub.3, --C(R)(J)(K) or --C(Z)(J)(K) are of particular
interest. Embodiments in which R.sup.7 and R.sup.8 are
independently H, ON, --NO.sub.2, halogen, J, -A-(M).sub.n
aliphatic, -G-(M).sub.n aliphatic, -(M).sub.n COCF.sub.3, (M).sub.n
OR, -(M).sub.n COOR, -A-(M).sub.n NRR', -G-(M).sub.q NRR',
-(M).sub.n CHO, -A-(M).sub.n N(R)(CO)R', -G-(M).sub.q N(R)(CO)R',
-A-(M).sub.n --CO--NRR, or G-(M).sub.n --CO--NRR, where the
aliphatic groups may be substituted or unsubstituted; or R.sup.7 is
a covalent bond to an R.sup.4 substituent of X to form a ring of 4
to 8 atoms are also of particular interest (including among others,
those compounds in which the R groups of R.sup.6 and/or of R.sup.7
and R.sup.8 are H). This set of compounds is illustrated by those
in which R.sup.6 comprises --OR, -APO.sub.3 RR', --OPO.sub.3 RR',
-ACO.sub.2 R, -ACOCF.sub.3 or C(R)(J)(K); A comprises -M.sub.m -
(e.g., --CH.sub.2 --, --CF.sub.2 --, --CHF--, --CHOH--, --CH.sub.2
CF.sub.2 --, etc), GM- (e.g. --OCH.sub.2 --) or a covalent bond;
each R and R' is H, or substituted or unsubstituted lower alkyl or
substituted or unsubstituted benzyl; and, R.sup.7 and R.sup.8 are
independently H, J, -A-(M).sub.n substituted or unsubstituted
aliphatic, -(M).sub.n COCF.sub.3, -(M).sub.n OH, -(M).sub.n COOR,
-A-(M).sub.n NRR', -(M).sub.n CHO, -A-(M).sub.n N(R)(CO)R' or
-A-(M).sub.n --CO--NRR. For example, in some such cases, R.sup.6
comprises --OH, -PO.sub.3 RR, OPO.sub.3 RR, --CH.sub.2 PO.sub.3 RR,
--CF.sub.2 PO.sub.3 RR, --OCH.sub.2 CO.sub.2 R, --NHCH.sub.2
CO.sub.2 R, --CH.sub.2 CO.sub.2 R, --CF.sub.2 CO.sub.2 R,
-N(PO.sub.3 RR')(PO.sub.3 RR')', --CH.sub.2 SO.sub.3 R, CF.sub.2
SO.sub.3 R, CH.sub.2 COCF.sub.3, --CF.sub.2 COCF.sub.3,
--CH(PO.sub.3 RR').sub.2 --CH(OH)(PO.sub.3 RR'),
--CH(NH.sub.2)(PO.sub.3 RR'), --CH (CO.sub.2 R).sub.2,
--CF(CO.sub.2 R).sub.2, --CH(PO.sub.3 R RR)(CO.sub.2 R"),
--CH(PO.sub.3 RR')(SO.sub.3 R"), --CH(PO.sub.3 RR')(SO.sub.2
NH.sub.2), --CH(SO.sub.2 NH.sub.2).sub.21 or --CH(SO.sub.3
RR).sub.2. In some such compounds, one or more of R, R' and R" in
the -PO.sub.3 RR', --OPO.sub.3 RR', --H.sub.2 PO.sub.3 RR',
--CF.sub.2 PO.sub.3 R, --OCH.sub.2 CO.sub.2 R, --NHCH.sub.2
CO.sub.2 R, --CH.sub.2 CO.sub.2 R, --CF.sub.2 CO.sub.2 R,
--N(PO.sub.3 RR')(PO.sub.3 RR')', --CH.sub.2 SO.sub.3 R, F.sub.2
SO.sub.3, --CH.sub.2 COCF.sub.3, --CF.sub.2 COCF.sub.3,
--CH(PO.sub.3 RR').sub.2, --CH(OH)(PO.sub.3 NRR),
CH(NH.sub.2)(PO.sub.3 RR), --CH(CO.sub.2 R).sub.2, --CF(CO.sub.2
R).sub.2, --CH(PO.sub.3 RR')(CO.sub.2 R"), --CH(PO.sub.3
RR')(SO.sub.3 R"), --CH(PO.sub.3 RR')(SO.sub.2 NH.sub.2),
--CH(SO.sub.2 NH.sub.2).sub.2, or --CH(SO.sub.3 RR').sub.2 moiety
is H. In others, one or more of those R groups is -(M).sub.m
--CH.sub.2 Z, -(M).sub.m --CHZ.sub.2, -(M).sub.m --CZ.sub.3,
--R.sup.15, -M--O--CO----OR.sup.15 or -M--O--CO--R.sup.15, where Z
is halogen and R.sup.15 is substituted or unsubstituted lower
aliphatic, aryl or heterocyclic. For example, in various
embodiments, R.sup.15 is methyl, ethyl, n-propyl, i-propyl,
n-butyl, isobutyl, tbutyl, n-amyl, sec-amyl, benzyl or substituted
benzyl, and M is CH.sub.2, CHR (e.g. CHCH.sub.3 etc.) and the
like.
Further illustrations include CH.sub.2 --O--CO--OEt,
--CH(Me)--O--CO--OEt, CH.sub.2 --O--CO-t-butyl, etc.
In one subgenus of the foregoing compounds, R.sup.7 and R.sup.8 are
both H. In another subgenus R.sup.7 is --N(M nCOOR)(MnCOOR)', e.g.,
--N(CH.sub.2 CO.sub.2 R).sub.2. In another subgenus, R.sup.7 is J,
-A-(M).sub.n (substituted or unsubstituted aliphatic, aryl or
heterocyclic), -(M).sub.n COC F.sub.3, -(M).sub.n OH, -(M).sub.n
COOR, -A-(M).sub.n NRR', -(M).sub.n CHO, -A-(M).sub.n N(R)(CO)R',
-A-(M).sub.n --NRR or-A-(M).sub.n --CO--NRR; and R.sup.8 is H. The
latter subgenus is illustrated by compounds in which R.sup.7 is
lower alkyl, lower alkenyl, --OH, --NH.sub.2, --NO.sub.2, --CN,
--NHR, --NHCOR, --CHO, --CH.sub.2 CHO, --PO.sub.3 RR, --OPO.sub.3
RR, --CH.sub.2 PO.sub.3 RR, CF.sub.2 PO.sub.3 RR, --OCH.sub.2
CO.sub.2 R, --NHCH.sub.2 CO .sub.2 R, CH.sub.2 CO.sub.2 R,
--CF.sub.2 CO.sub.2 R, --SO.sub.3 R, CH.sub.2 SO.sub.3 R, CF.sub.2
SO.sub.3 R, COCF.sub.3, COCH.sub.2 F, COCF.sub.2 H, --CF.sub.2
COCF.sub.3 or--SO.sub.2 NH.sub.2. In some such compounds, one or
both of R and R' in --PO.sub.3 RR, PO RR, CH.sub.2 PO.sub.3 RR, 2
PO.sub.3 RR, --OCH.sub.2 CO.sub.2 R, --NHCH.sub.2 CO.sub.2 R,
--CH.sub.2 CO.sub.2 R, CF.sub.2 CO.sub.2 R, --SO.sub.3 R,
--N(PO.sub.3 RR')(PO.sub.3 RR')',--CH.sub.2 SO.sub.3 R, or CF.sub.2
SO.sub.3 R is H. In others, one or more of those R groups is
-(M).sub.m --CH.sub.2 Z, -(M).sub.m --CH.sub.2 Z, -(M).sub.m
CZ.sub.3, --R.sup.15, -M--O--CO--OR.sup.15 or -M--O--CO--R.sup.15,
where Z is halogen and R.sup.15 is substituted or unsubstituted
lower aliphatic, aryl or heterocyclic. For example, in individual
cases, R.sup.15 is methyl, ethyl, n-propyl, i-propyl, n-butyl,
isobutyl, tbutyl, n-amyl, sec-amyl, benzyl or substituted benzyl,
and M is CH.sub.2, CHR (e.g. CHCH.sub.3 etc.) and the like.
In an illustrative subgenus, R.sup.6 comprises -APO.sub.3 RR'
(e.g., --OPO.sub.3 H.sub.2) and R.sup.7 is -A-(M).sub.n substituted
or unsubstituted aliphatic.
In another subgenus, R.sup.6 and R.sup.7 are independently selected
from J and K.
In another subgenus, R.sup.6 is --C(R)(J)(K). Illustrative
compounds of this subgenus include those in which R.sup.6 is
--CH(J)(K) and those in which R.sup.6 is --C(R)(PO.sub.3 R'R")(K).
The latter compounds are illustrated by embodiments in which none,
one, two or three of the R groups of the --C(R)(PO.sub.3 R'R')(K)
moiety are H.
As in previously mentioned cases, compounds of this invention which
contain a moiety J, e.g., compounds of Formula I in which R.sup.6
is --C(R)(J)(K), include among others embodiments in which one or
both of R and R' (e.g., of a --PO.sub.3 RR moiety) are R.sup.15,
-(M).sub.m --CH.sub.2 Z'-(M).sub.m --CHZ.sub.2 -(M)M-CZ.sub.3,
-M--O--CO--OR.sup.15 or -M--O--CO--R.sup.15, where Z is halogen and
R.sup.15 is substituted or unsubstituted lower aliphatic, aryl or
heterocyclic (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl,
isobutyl, t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl),
and M is CH.sub.2, CHR (e.g. CHCH.sub.3 etc.) and the like.
The compounds of Formula I, including the various subgenera and
illustrative examples described above, all contain a bicyclic
moiety, B, as that term is defined herein and as is illustrated by
the following formula: ##STR16##
Compounds of this invention include those in which each of R.sup.9,
R.sup.10, R.sup.11 and R.sup.12 is independently 5M).sub.n Z,
dM).sub.n R, G(M).sub.n R, (M).sub.n WR or -(M).sub.n -W-GR. In
certain embodiments, one or more of the R, R' and R" groups of
R.sup.9, R.sup.10, R.sup.11, and R.sup.12 comprise a halo, hydroxy,
aliphatic, amino, amido or sulfonamido moiety. In some embodiments,
one or more of R.sup.9, R.sup.10, R.sup.11, and R.sup.12 is a
substituted aliphatic moiety containing at least one substituent
selected from substituted or unsubstituted cycloaliphatic,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, --COR, --CO.sub.2 R, --CONRR', and --OR. In some
embodiments of particular interest, one or more of R.sup.9,
R.sup.10, R.sup.11, and R.sup.12 comprises -(M).sub.n
(cycloaliphatic), -(M).sub.n (substituted or unsubstituted aryl),
-(M).sub.n (substituted or unsubstituted heteroaryl), -(M).sub.n
CHO, HM).sub.n CONH.sub.2, -(M).sub.n CSNH.sub.2, -(M).sub.n
SONH.sub.2, -(M).sub.n SO.sub.2 NRR', -(M).sub.n OR, -(M).sub.n
(lower aliphatic), -(M).sub.n -C(OR)R'R", or -(M).sub.n
--C.dbd.CRR'. For example, in some cases, one or more of R.sup.9,
R.sup.10, R.sup.11.sub.1 and R.sup.12 comprise methyl,
--CH.sub.2).sub.q R.sup.13 where q is 143 and R.sup.13 comprises
methyl; i-propyl; i-butyl; t-butyl; cycloaliphatic; phenyl;
substituted phenyl; naphthyl; substituted naphthyl; a 5, 6 or
7-membered heterocyclic ring or a bicyclic heterocylic moiety. In
some cases, R1.sup.2 comprises a formyl group on a ring nitrogen.
Possible substituents on the R, R' and R" groups include, among
others, halo, hydroxy, alkyl, amino, amido and sulfonamido
moieties. Other potential substituents are as disclosed elsewhere
herein, including in the numerous specific examples.
Other compounds of Formula I which are of particular interest
include those in which one or more of R.sup.9, R.sup.10, R.sup.11,
and R.sup.12 comprises -G(M).sub.n (aliphatic), G(M).sub.n
(cycloaliphatic), G(M).sub.n (substituted or unsubstituted aryl),
G(M).sub.n (substituted or unsubstituted heteroaryl), G(M).sub.n
CHO, G(M).sub.n CONH.sub.2, G(M).sub.n CSNH.sub.2, -G(M).sub.n
SONH.sub.2, G(M).sub.n SO.sub.2 NRR', -G(M).sub.n OR, G(M).sub.n
(lower aliphatic), -G(M).sub.n C(OR)R'R", or G(M).sub.n
--C.dbd.CRR', for instance as illustrated by cases in which
G(M).sub.n comprises --OCH.sub.2 --, --SCH.sub.2 -- or --NRCH.sub.2
--.
Compounds of particular interest further include those in which one
or more of R.sup.9, R.sup.10, R.sup.11, and R.sup.12 comprises -(M)
W--NH--R, e.g., -(M).sub.n (CO)--NH--R, as illustrated by
--(CH.sub.2).sub.m CONH--R and G(O)NR, for example). In some
compounds one or more of R.sup.9, R.sup.10, R.sup.11, and R.sup.12
comprises --O(M).sub.m (aliphatic).
In some cases R.sup.11 and R.sup.12 are both H, as illustrated by
the following structure: ##STR17##
In illustrative embodiments, R.sup.5 comprises a substituted or
unsubstituted lower aliphatic moiety, R.sup.9 comprises -(M).sub.n
W--NH--R" and R.sup.10 comprises --O(M).sub.m (aliphatic). For
example, in some such compounds, R.sup.9 comprises --CONH--R" and
R.sup.10 comprises -OM-cycloaliphatic or -OM-branched chain
aliphatic. In other cases, R.sup.9 comprises --CH.sub.2 CONH--R and
R.sup.10 comprises -OM-cycloaliphatic or -OM-branched chain
aliphatic.
From the perspective of Y moieties, compounds of interest include
those compounds of Formula I, including those of the various
subgenera and examples herein, in which Y comprises ##STR18##
particularly where R.sup.6 comprises --OR', -APO.sub.3 R'R",
--ASO.sub.3 R', -ACO.sub.2 R', --ASO.sub.2 NR'R", -ACOCF.sub.3, or
--C(R')(J)(K); and, R.sup.7 is H, --CN, --NO.sub.2, halogen, J,
-A-(M).sub.n substituted or unsubstituted aliphatic, -(M).sub.n
COCF.sub.3, -(M).sub.n OH, -(M).sub.n COOR, -A-(M).sub.n NRR',
-(M).sub.n CHO, -A-(M).sub.n N(R)(CO)R' or -A-(M).sub.n --CO--NRR'.
For example, in some cases R.sup.6 comprises --PO.sub.3 RR',
--OPO3RR', --OSO.sub.2 NRR', --(CH.sub.2)PO.sub.3 RR',
--(CF.sub.2)PO.sub.3 RR' or GCRJK; and R.sup.7 comprises R
(including among others, H, alkyl, alkenyl, etc.) --CN, amido,
acylamino, J (e.g. --CO.sub.2 R), or --CHO. For example, in some
cases, R.sup.6 comprises --OPO.sub.3 RR' or --CF.sub.2)PO.sub.3 RR'
and R.sup.7 is H. In some embodiments one or more R groups
(including R', R", etc) of R.sup.6 comprises -(M).sub.m CH.sub.2 Z,
-(M).sub.m CHZ.sub.2, -(M).sub.m --CZ.sub.3, --R.sup.15,
-M--O--CO--OR.sup.15 or -M--O--CO--R.sup.15, where Z is H or
halogen and R.sup.15 is substituted or unsubstituted lower
aliphatic, aryl or heterocyclic. For example, in individual cases,
R.sup.15 is methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl,
t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl, and M is
CH.sub.2, CHR (e.g. CHCH.sub.3 etc.) and the like.
Compounds of the Structure ##STR19##
(as well as homologs in which p is 1 or 2) are of interest as
intermediates in the preparation of compounds of this invention. Of
particular interest are such compounds in which R.sup.9 and
R.sup.10 are independently halo, R, --OR, --SR, --NRR', --COR, or
-(M).sub.n W-NHR, and R.sup.11 and R.sup.12 are as previously
defined, including cases in which R.sup.11 and R.sup.12 are H.
Compounds of this invention which are of special interest include
those which bind to a given SH2 domain (or protein containing such
SH2 domain) with a IC.sub.50 value of less than 50 .mu.M,
preferably less than 20 .mu.M, as determined by any scientifically
valid method, in vitro or in vivo. SH2 domains of current interest
include those of a Src, Fyn, Lck, Yes, BIk, Lyn, Fgr, Hck, Yrk,
ZAP-70, Syk, STAT or AbI protein.
Also of interest are pharmaceutical compositions comprising a
compound of this invention, or a pharmaceutically acceptable
derivative thereof, and one or more pharmaceutically acceptable
excipients.
Compounds of this invention (or a composition containing such a
compound) can be administered to cells or to animals, preferably a
mammal in need thereof, as a method for inhibiting SH2-mediated
signal transduction therein. In particular cases, it will be
advantageous to carry out that method using a pharmaceutical
composition containing a compound which specifically binds to an
SH2 domain of Src, ZAP-70, Syk, or STAT 6. In other cases it will
be advantageous to carry out that method where the SH2-mediated
signal transduction is mediated by a PDGF receptor protein, EGF
receptor protein, HER2tNeu receptor protein, fibroblast growth
factor receptor protein, focal adhesion kinase protein, p130
protein, or p68 protein.
Cases in which a mammal may be in need of inhibition of
SH2-mediated signaling include cases in which the mammal has a
proliferative disease, cancer, restenosis, osteoporosis,
inflammation, allergies, or cardiovascular disease. In such cases,
administering a therapeutically effective amount of the composition
to the mammal, preferably to a human patient, will constitute
treating or preventing the proliferative disease, cancer,
restenosis, osteoporosis, inflammation, allergic reaction, or
cardiovascular disease in the recipient or a method for causing
immunosuppression in the recipient.
Generally preferred compounds of this invention include any of the
foregoing compounds which yield an observable IC.sub.50 value, when
tested against an SH2 domain of interest and a pTyr-containing
peptide ligand (or mimic thereof) for that SH2 domain, of 50 .mu.M
or better, preferably 5 .mu.M or better, more preferably 1 .mu.M or
better, and even more preferably, 500 nM or better, as determined
by any scientifically valid measure, especially when the SH2 domain
is from a Src, Fyn, Lck, Yes, Blk, Lyn, Fgr, Hck, Yrk, ZAP, Syk,
STAT or AbI protein.
A pharmaceutical composition may be prepared containing a compound
of this invention (including a pharmaceutically acceptable
derivative thereof) together with one or more pharmaceutically
acceptable excipients.
A compound of this invention, preferably in the form of a
pharmaceutical composition, may be administered to a mammal in need
thereof, preferably a human patient, as a method for inhibiting
SH2-mediated signal transduction in the recipient mammal. In some
cases, the compound may be selected based on its ability to
specifically bind to an SH2 domain, e.g. of Src, ZAP-70, Syk, or
STAT 6, etc., or on its ability to inhibit a signal transduction
pathway mediated by an SH2 domain-containing protein. Such use of
an appropriately selected compound of this invention thus provides
a method for inhibiting SH2-mediated signal transduction which is
mediated by a PDGF receptor protein, EGF receptor protein, HER2/Neu
receptor protein, fibroblast growth factor receptor protein, focal
adhesion kinase protein, p130 protein, or p68 protein. Use of a
compound of this invention may be particularly advantageous in
cases in which the mammal has a proliferative disease, cancer,
restenosis, osteoporosis, inflammation, allergies, or
cardiovascular disease. In such cases, administering to the patient
a therapeutically effective amount of a compound of this invention,
preferably in the form of a pharmaceutical composition, provides a
method for treating or preventing a proliferative disease, cancer,
restenosis, osteoporosis, inflammation, allergies, or
cardiovascular disease in the patient.
DETAILED DESCRIPTION OF THE INVENTION
Compounds and Definitions
As mentioned above, this invention provides a novel class of
compounds useful as inhibitors of signal transduction pathways
mediated by the interaction of protein receptors for
phosphotyrosine-containing proteins, such as proteins containing
one or more SH2 domains, with their phosphotyrosine-containing
ligands. Compounds of this invention comprise those of Formula I,
set forth above, and are illustrated in part by the various
classes, subgenera and subsets of compounds noted above, and by the
various subgenera and species disclosed elsewhere herein. The
compound may be in the form of an individual enantiomer,
diastereomer or geometric isomer, or may be in the form of a
mixture of stereoisomers.
Also included are pharmaceutically acceptable derivatives of the
foregoing compounds, where the phrase "pharmaceutically acceptable
derivative" denotes any pharmaceutically acceptable salt, ester, or
salt of such ester, of such compound, or any other adduct or
derivative which, upon administration to a patient, is capable of
providing (directly or indirectly) a compound as otherwise
described herein, or a metabolite or residue thereof, preferably
one which is a signal transduction inhibitor. Pharmaceutically
acceptable derivatives thus include among others pro-drugs. A
pro-drug is a derivative of a compound, usually with significantly
reduced pharmacological activity, which contains an additional
moiety which is susceptible to removal in vivo yielding the parent
molecule as the pharmacologically active species. An example of a
pro-drug is an ester which is cleaved in vivo to yield a compound
of interest. Pro-drugs of a variety of compounds, and materials and
methods for derivatizing the parent compounds to create the
pro-drugs, are known and may be adapted to the present
invention.
The term "aliphatic" as used herein includes both saturated and
unsaturated, straight chain (i.e., unbranched), branched, cyclic,
or polycyclic aliphatic hydrocarbons, which are optionally
substituted with one or more functional groups. Unless otherwise
specified, alkyl, other aliphatic, alkoxy and acyl groups
preferably contain 1-8, and in many cases 1-6, contiguous aliphatic
carbon atoms. Illustrative aliphatic groups thus include, for
example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl,
--CH.sub.2 -cyclopropyl, allyl, n-butyl, sec-butyl, isobutyl,
tert-butyl, cyclobutyl, --CH.sub.2 -cyclobutyl, n-pentyl,
sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, --CH.sub.2
-cyclopentyl, n-hexyl, sec-hexyl, cycycloxyl, --CH.sub.2
-cycycloxyl moieties and the like, which again, may bear one or
more substituents.
Some examples of substituents of aliphatic (and other) moieties of
compounds of this invention include: R, --OH, --OR, --SH,
--SR,--CHO, .dbd.O, --COR, --COOH (or amide, ester, carbamate,
urea, oxime or carbonate thereof), --NH.sub.2 (or substituted
amine, amide, urea, carbamate or guanidino derivative therof),
halo, trihaloalkyl, cyano, --SO.sub.2 --CF.sub.3, --OSO.sub.2 F,
--OS(O).sub.2 R, --SO.sub.2 --NHR, --NHSO.sub.2 R, sulfate,
sulfonate, aryl and heteroaryl moieties. Aliphatic, heteraliphatic,
aryl and heterocyclic substituents may themselves be substituted or
unsubstituted (e.g. mono-, di- and tri-alkoxyphenyl;
methylenedioxyphenyl or ethylenedioxyphenyl; halophenyl; or
-phenyl-G(Me).sub.2 --CH.sub.2 --O--CO--[C3-C6] alkyl or
alkylamino). Additional examples of generally applicable
substituents are illustrated by the specific embodiments shown in
the Examples which follow.
The term "aliphatic" is thus intended to include alkyl, alkenyl,
alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties.
As used herein, the term "alkyl" includes both straight, branched
and cyclic alkyl groups. An analogous convention applies to other
generic terms such as "alkenyl", "alkynyl" and the like.
Furthermore, as used herein, the language "alkyl", "alkenyl",
"alkynyl" and the like encompasses both substituted and
unsubstituted groups.
The term "alkyl" refers to groups usually having one to eight,
preferably one to six carbon atoms. For example, "alkyl" may refer
to methyl, ethyl, n-propyl, isopropyl, cyclopropyl, butyl,
isobutyl, sec-butyl, tert-butyl, cyclobutyl, pentyl, isopentyl
tert-pentyl, cyclopentyl, hexyl, isohexyl, cycycloxyl, and the
like. Suitable substituted alkyls include, but are not limited to,
fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl,
3-fluoropropyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl,
benzyl, substituted benzyl and the like.
The term "alkenyl" refers to groups usually having two to eight,
preferably two to six carbon atoms. For example, "alkenyl" may
refer to prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl,
hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, and the like. The
language "alkynyl," which also refers to groups having two to
eight, preferably two to six carbons, includes, but is not limited
to, prop-2-ynyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl,
3-methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl, and the like.
The term "cycloalkyl" as used herein refers specifically to groups
having three to seven, preferably three to ten carbon atoms.
Suitable cycloalkyls include, but are not limited to cyclopropyl,
cyclobutyl, cyclopentyl, cycycloxyl, cycloheptyl and the like,
which, as in the case of other aliphatic or heteroaliphatic or
heterocyclic moieties, may optionally be substituted.
The term "heteroaliphatic" as used herein refers to aliphatic
moieties which contain one or more oxygen, sulfur, nitrogen,
phosphorous or silicon atoms, e.g., in place of carbon atoms.
Heteroaliphatic moieties may be branched, unbranched or cyclic and
include heterocycles such as morpholino, pyrrolidinyl, etc.
The term "heterocycle" as used herein refers to cyclic
heteroaliphatic and heteroaryl groups and preferably three to ten
ring atoms total, includes, but is not limited to heteroaliphatic
moieties such as oxetane, tetrahydrofuranyl, tetrahydropyranyl,
aziridine, azetidine, pyrrolidine, piperidine, morpholine,
piperazine and the like, and heteroaryl moieties as described
below.
The terms "aryl" and "heteroaryl" as used herein refer to stable
mono- or polycyclic, heterocyclic, polycyclic, and polyheterocyclic
unsaturated moieties having 3-14 carbon atom which may be
substituted or unsubstituted. Substituents include any of the
previously mentioned substituents. Non-limiting examples of useful
aryl ring groups include phenyl, halophenyl, alkoxyphenyl,
dialkoxyphenyl, trialkoxyphenyl, alkylenedioxyphenyl, naphthyl,
phenanthryl, anthryl, phenanthro and the like. Examples of typical
heteroaryl rings include 5-membered monocyclic ring groups such as
thienyl, pyrrolyl, imidazolyl, pyrazolyl, furyl, isothiazolyl,
furazanyl, oxazolyl, isoxazolyl, thiazolyl, oxadiazolyl and the
like; 6-membered monocyclic groups such as pyridyl, pyrazinyl,
pyrimidinyl, pyridazinyl, triazinyl and the like; and polycyclic
heterocyclic ring groups such as benzo[b]thienyl,
naphtho[2,3-b]thienyl, thianthrenyl, isobenzofuranyl, chromenyl,
xanthenyl, phenoxathienyl, indolizinyl, isoindolyl, indolyl,
indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl,
naphthyridinyl, quinoxalinyl, quinazolinyl, benzothiazole,
benzimidazole, tetrahydroquinoline cinnolinyl, pteridinyl,
carbazolyl, beta-carbolinyl, phenanthridinyl, acridinyl,
perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl,
phenothiazinyl, phenoxazinyl, and the like(see e.g. Katritzky,
Handbook of Heterocyclic Chemistry). The aryl or heteroaryl
moieties may be substituted with one to five members selected from
the group consisting of hydroxy, C1-C8 alkoxy, C1-C8 branched or
straight-chain alkyl, acyloxy, carbamoyl, amino, N-acylamino,
nitro, halo, trihalomethyl, cyano, and carboxyl. Aryl moieties thus
include, e.g. phenyl; substituted phenyl bearing one or more
substituents selected from groups including: halo such as chloro or
fluoro, hydroxy, C1-C6 alkyl, acyl, acyloxy, C1-C6 alkoxy (such as
methoxy or ethoxy, including among others dialkoxyphenyl moieties
such as 2,3-, 2,4-, 2,5-, 3,4- or 3,5-dimethoxy or diethoxy phenyl
or such as methylenedioxyphenyl, or 3-methoxy-5-ethoxyphenyl; or
trisubstituted phenyl, such as trialkoxy (e.g., 3,4,5-trimethoxy or
ethoxyphenyl), 3,5-dimethoxy-4-chloro-phenyl, etc.), amino,
--SO.sub.2 NH.sub.2, --SO.sub.2 NH(aliphatic), --SO.sub.2
N(aliphatic).sub.2, --O-aliphatic-COOH, and --O-aliphatic--NH.sub.2
(which may contain one or two N-aliphatic or N-acyl
substituents).
A "halo" substituent may be fluoro, chloro, bromo or iodo.
With respect to nomenclature, note that asymetric moieties such as
"G-M-" are written in the direction or order in which they are
intended to be read into a given structure. Thus, "-G-M-" is
distinct from "-M-G." For example, in "Ar-A-COOR", where A is
-G-M-, the structure Ar--G-M--COOR, not Ar--M-G--COOR, is
intended.
Synthesis
Those of ordinary skill in this art will appreciate that compounds
of this invention may be produced using any of a variety of
synthetic strategies. We typically use a convergent synthetic
scheme in which an intermediate comprising the desired "YXU"
moiety, protected as appropriate, is condensed with a second
intermediate comprising the desired amino moiety HR.sup.14
N(CR.sup.1 R.sup.2).sub.m B, again, protected as appropriate, to
yield (following any necessary deprotection steps) the desired
compound of Formula I. A variety of methods and materials for
effecting the relevant chemical transformations, product recovery,
purification and formulation are known in the art which may be
adapted to use in the practice of this invention. The detailed
examples which follow illustrate such syntheses and should provide
helpful guidance to the practitioner.
Assays for Comparative Functional Evaluation of Compounds
Compounds of this invention may be evaluated in a variety of assays
to determine their relative ability to bind to a receptor for a
pTyr-containing ligand, such as a protein containing one or more
SH2 or PI domains, or to otherwise inhibit an intermolecular
interaction mediated by such a domain. See e.g. U.S. Pat. No.
5,667,980 (Pawson; competitive binding assays), PCT/US97/02635
(Rickles et al; cell-based assays) and PCT/US97/06746 (Lynch et al,
FP assays). Compounds may also be evaluated for their selectivity
of binding to one such receptor (or family of receptors) relative
to another such receptor (or family of receptors). The compounds of
this invention can be further evaluated by conventional methods for
possible therapeutic applications, including evaluations of
toxicological and pharmacological activity. For example, the
compounds may further be evaluated for activity in inhibiting
cellular or other biological events mediated by a pathway involving
the molecular interaction of interest using a suitable cell-based
assay or an animal model. Cell-based assays and animal models
suitable for evaluating inhibitory activity of a test compound with
respect to a wide variety of cellular and other biological events
are known in the art. New assays and models are regularly developed
and reported in the scientific literature.
By way of non-limiting example, compounds which bind to an SH2
domain involved in the transduction of a signal leading to asthma
or allergic episodes may be evaluated in a mast cell or basophil
degranulation assay. The inhibitory activity of a test compound
identified as an SH2 inhibitor by the method of this invention with
respect to cellular release of specific mediators such as
histamine, leukotrienes, hormonal mediators and/or cytokines, as
well as its biological activity with respect to the levels of
phosphatidylinositol hydrolysis or tyrosine phosphorylation can be
characterized with conventional in vitro assays as an indication of
biological activity. [See, e.g., Edward L. Barsumian et al, Eur. J.
Immunol., 11:317-323 (1981); M. J. Forrest, Biochem. Pharmacol.,
42:1221-1228 (1991) (measuring N-acetyl-betaglucosamin-adase from
activated neutrophils); and Stephan et al., J. Biol. Chem.,
7:5434-5441 (1992)].
For example, histamine release can be measured by a
radioimmunoassay using a kit available from AMAC Inc. (Westbrook,
Me.). One can thus evaluate the biological activity of compounds of
this invention and compare them to one another and to known active
compounds or clinically relevant compounds which can be used as
positive controls.
Generally speaking, in such assays IC50 scores of 20 .mu.M or less
are considered of special interest, scores below 1 .mu.M are
considered of particular interest and scores below about 500 nM are
of high interest. Inhibitors of this invention may also be tested
in an ex vivo assay, e.g., for their ability to block
antigen-stimulated contraction of sensitized guinea pig tracheal
strip tissue. Activity in this assay has been shown to be useful in
predicting the efficacy of potential anti-asthma drugs.
Numerous animal models of asthma have been developed and can be
used [for reviews, see Larson, "Experimental Models of Reversible
Airway Obstruction", in THE LUNG, Scientific Foundations, Crystal,
West et al. (eds.), Raven Press, New York, pp. 953-965 (1991);
Warner et al., Am. Rev. Respir. Dis., 141:253-257 (1990)]. Species
used in animal models of asthma include mice, rats, guinea pigs,
rabbits, dogs, sheep and primates. Other in vivo models available
are described in Cross et al., Lab Invest., 63:162-170 (1990); and
Koh, et al., Science, 256:1210-1213 (1992).
By way of further example, compounds which bind to an SH2 or other
domain of interest involved in the transduction of a signal
involved in the initiation, maintenance or spread of cancerous
growth may be evaluated in relevant conventional in vitro and in
vivo assays. See e.g., Ishii et al., J. Antibiot., XLII:1877-1878
(1989); and U.S. Pat. No. 5,206,249 (issued Apr. 27, 1993).
Compounds which bind to a ZAP SH2 domain or which otherwise inhibit
ZAP-70-mediated signaling may be evaluated for immunosuppressive
activity, e.g., in any of the well-known in vitro or in vivo
immunosuppression assays.
Compounds which bind to a Src SH2 domain or which otherwise inhibit
Src-mediated signaling may be evaluated for activity in a variety
of assays considered predictive of activity in treating or
preventing osteoporosis. Such assays include the various pit assays
and calvaria assays, among others. Illustrative assays are
described below.
Murine Calvaria Assay
In osteoporosis, excessive bone resorption results in decreased
bone density. In vivo and in vitro models of bone resorption are
used to study the processes leading to osteoporosis. In vitro,
fetal rat long bone and murine calvaria cultures are routinely
used. Both models display similar responses to parathyroid hormone
(PTH), a physiological modulator of bone resorption (Stem, P. H.
and N. S. Krieger. Comparison of fetal rat limb bones and neonatal
mouse calvaria: effects of parathyroid hormone and
1,25-dihydroxyvitamin D.sub.3. Calcif. Tissue Int. 35: 172-176,
1983). The calvaria model of bone resorption can be successfully
used to screen osteotropic compounds as has been previously shown
(Green, J. R., K. Muller and K. Jaeggi. Preclinical pharmacology of
CGP 42'446, a new, potent, heterocyclic bisphosphonate compound. J.
Bone Miner. Res. 9: 745-751, 1994.).
In one modification of the conventional calvaria model, calvaria
are not labeled with .sup.45 Ca.sup.++. Instead, calvarial calcium
release into the media is assessed using a microtiter colorimetric
calcium assay. This modification can yield more consistent
responses than the radioactive methodology and provides results
which are comparable to literature values for .sup.45 Ca.sup.++
assays.
One calvaria culture model tests the ability of anti-resorptive
compounds to prevent resorption (prophylactic model). A second
model tests the ability of these compounds to terminate ongoing
resorption (therapeutic model). Cytotoxicity may be assessed in
both models using a lactate dehydrogenase (LDH) assay. These in
vitro models of bone resorption may be used for routine screening
and evaluation of compounds for their ability to alter
osteoclast-mediated bone resorption.
Media Preparation
Calcium free Dulbecco's Modified Eagle's Medium (DMEM) may be
obtained in a 5.times.solution (Specialty Media, D-012). A
1.times.solution is prepared using ultrafiltered water. A suitable
media contains 15% heat inactivated horse serum (Sigma, H 1270).
Calcium concentration is adjusted to 1.65 to 1.83 mM using 0.2 M
CaCl.sub.2. Penicillin (100 U/ml) and streptomycin (0.1 mg/ml) are
added to the final media preparation. Indomethacin is prepared to
0.5 mg/ml (1.397.times.10.sup.-7 M) in ethanol, and is added to an
aliquot of DMEM to produce a final concentration of 0.5 .mu.M.
Bovine parathyroid hormone (1-34) may be obtained from Bachem (PCAL
100). PTH is solubilized in 0.1% BSA and is then diluted in DMEM to
produce a final concentration of 10-6 M PTH. Ten-fold serial
dilutions are performed down to 10.sup.-11 M.
Calvaria Dissection
Pregnant CD-1 mice may be obtained from Charles River and are
subjected to parturition. Neonatal mice (4-6 days) are cleansed
with betadine and then euthanized by decapitation. Adherent skin is
cleared away from the skull, exposing the calvaria. The calvaria
are dissected away from the skull using a 12B scalpel. Calvaria are
immediately placed into a glass petri dish containing room
temperature Tyrode's Salt Solution (Sigma, T-2397). The calvaria
are trimmed free of cartilage and bisected with a scalpel along the
sagital suture. After dissection of all calvaria, calvaria are
transferred into 24 well plates containing 0.5 .mu.M indomethacin
(Sigma, 1-7378).
Culture Conditions
Calvaria are incubated in 1.5 ml DMEM in 24 well tissue culture
plates at 37.degree. C., 5% CO/air. Plates are rocked in the
incubator using a Bellco rocker platform. Calvaria are
pre-incubated in 0.5 .mu.M indomethacin for 24 hours. For each
experiment, 6 to 8 random calvaria halves are used for each group.
Both halves from a single mouse are never in the same group.
Experiments are repeated at least three times.
Prophylactic Calvadia Experiment
After the 24 h pre-incubation period, calvaria are thoroughly
washed in indomethacin-free DMEM. Calvaria are then transferred to
new wells containing various PTH concentrations, and are cultured
for an additional 72 hours. Media samples (30 .mu.l) are obtained
every 24 hours and assayed for calcium and LDH activity.
Therapeutic Calvaria Experiment
At the end of the 24 h pre-incubation period, the calvaria are
washed free of indomethacin using DMEM. Calvaria are then
transferred to new wells containing DMEM or various concentrations
of PTH. After 24 hours calvaria are transferred into new wells with
fresh media (PTH or DMEM) and cultured an additional 48 hours
before addition of control vehicle. This may be accomplished by
adding 3 .mu.l of DMSO to new wells, and transferring each calvaria
along with its media into wells. Culture continues for a further 24
hours. Media samples are obtained after 72 hours and 96 hours in
culture with PTH and assayed for calcium. Additional samples are
obtained after 48, 72, and 96 hours in culture with PTH and assayed
for LDH.
Calcium Assay
A commercially available diagnostic calcium assay (Sigma, No.
588-3), modified for use in a microtiter format, may be used to
determine circulating serum calcium concentrations. This
colorimetric assay is dependent on the specific, high affinity
complexation of calcium with arsenazo III dye under acidic
conditions, which occurs with 1:1 stoichiometry and absorbs at 600
nm (Bauer, P. J. Affinity and stoichiometry of calcium binding by
Arsenazo III. Anal Biochem, 110:61, 1981; Michaylova, V and P
Ilkova. Photometric determination of micro amounts of calcium with
Arsenazo III. Anal Chim Acta, 53: 194, 1971). Magnesium has very
low affinity for arsenazo III.
Briefly, 15 p1 of media or rat sera (see below) is diluted 18-fold
with ultrafiltered water (nearly calcium-free). Fifty p, of this
solution are pipetted into microtiter wells (Nunc, Maxisorp,
flat-bottom, 0.4 ml/well). Standards of 0, 0.5, 1, 2.5, 3.75, 5,
6.25, and 7.5 mg/dl (mg %) calcium, diluted 8-fold with
ultrafiltered water from control standards (Sigma, 360-11), are
used to construct standard curves. Once all standards and samples
are pipetted onto the plate, 150 .mu.l of diagnostic reagent is
added to initiate complexation. Optical density measurements are
obtained on a microtiter plate reader (Molecular Devices,
ThermoMax) at 600 nm.
Lactate Dehydrogenase Assay
Phosphate buffer is prepared in distilled water (0.26 M K.sub.2
HPO.sub.4.multidot.3H.sub.2 O, 0.26 M KH.sub.2 PO.sub.4 ; pH 7.4).
A mix consisting of: 22 ml of phosphate buffer, 6 ml distilled
water and 2.0 ml of 0.01 M pyruvate is prepared. NADH is prepared
to 0.4 mg/ml in phosphate buffer.
Ten .mu.l of media samples obtained from incubated calvaria are
added to 96 well plates. Wells containing 10 .mu.l of DMEM serve as
blanks. To each well, 90 .mu.l distilled water and 150 .mu.l
phosphate mix is added. 50 .mu.l NADH is added using an eight
channel pipette immediately before the plate is read on a
microtiter plate reader at 340 nm. A kinetic assay is performed for
10 minutes, with a read interval of 20 seconds.
Thyroid/Parathyroidectomized Rat Model Of Bone Resorption
Parathyroid hormone (PTH) replacement in thyroparathyroidectomized
(TPTX) rats is routinely used as an in vivo model of controlled
bone resorption. Rats are the species of choice since the
mechanisms of bone modeling in the rat resemble those in humans. In
addition, hormones and pharmacologic agents have similar effects on
both rat and human bone (Frost, H. M. and W. S. S. Jee. On the rat
model of human osteopenias and osteoporoses. Bone and Mineral, 18:
227-236, 1992). Removal of the thyroid and parathyroid glands
results in a rapid loss of parathyroid hormone (PTH) from the
circulation. Since PTH induces osteoclast-mediated bone resorption,
this process is inhibited in TPTX animals. In addition, PTH
mediates calcium reabsorption from the kidneys and absorption from
the small intestines. The lack of these activities work in concert
to decrease serum calcium levels. In the absence of PTH, rats
remain in a hypocalcemic state. Restriction of dietary calcium
limits intestinal calcium absorption and renal calcium filtration
such that serum calcium levels are primarily influenced by bone
resorption. Controlled PTH replacement therapy results in a
controlled return of serum calcium to baseline levels. When
replacement occurs, concomitantly with a low calcium diet, serum
calcium increase is due to PTH-induced osteoclast-mediated bone
resorption. In this model, drugs which inhibit bone resorption
prevent the PTH-mediated return of serum calcium to baseline
levels.
Female Wistar rats (226-250 gm, Charles River) are fasted overnight
and anesthetized with 0.15 ml of 1.2% tribromoethanol (TBE). The
ventral neck area is shaved and swabbed with betadine and
isopropanol. A midline incision is made in the neck through the
skin and superficial muscle layer, as well as in the sternohyoid
muscle. Blunt dissection is performed to expose the thyroid gland.
The thyroid gland is carefully isolated from the trachea,
thyrohyoid muscle, as well as adjacent nerves and blood vessels,
using blunt dissection. The thyroid gland is excised one lobe at a
time. Cautery is performed for hemostasis. Care is taken to avoid
damaging the recurrent laryngeal nerve since damage to it is shown
to affect serum calcium concentrations (Hirsch, P. F., G. F.
Gauthier and P. L. Munson. Thyroid hypocalcemic principle and
recurrent laryngeal nerve injury as factors affecting the response
to parathyroidectomy in rats. Endocrinology, 73:244-252, 1963. et
al., 1963). The incisions are closed using 3-0 vicryl. The wound is
coated with triple antibiotic ointment (Fougera; 400 units/g
bacitracin zinc, 5 mg/g neomycin sulfate, 5000 units/g polymyxin B
sulfate). Following TPTX, rats are pair fed a low calcium diet
(Harlan Teklad TD 95065; 1).003% Ca.sup.++, .ltoreq.0.04% PO.sub.4)
such that each rat receives the same quantity of food. Rats are fed
at least 5 grams, but not more than 10 grams, of food. Rats
consuming less than 3.0 grams of food receive the nutritional
supplement Nutri-Cal p.o. (Evsco; .ltoreq.0.0033% calcium).
PTH Dose Response/Pump Implantation
Three days post TPTX, rats which are found to be hypocalcemic,
based on day 2 serum calcium levels, are implanted with
PTH-containing Alzet mini-osmotic pumps (ALZA, model 2001 D) which
pumps at a rate of 1 .mu.l/h. The rats are anesthetized with
ketamine (50 mg/kg, i.p.) and acepromazine (1.67 mg/kg, i.p.). The
scapula region is shaved and prepared for surgery with betadine and
isopropanol. A lateral incision of approximately 2 cm in length is
made between the scapulae. Using hemostats, a subcutaneous pocket
is created into which the Alzet pump is inserted. The wound is
closed either with nylon suture or with staples. Triple antibiotic
ointment is applied as described previously.
Bovine parathyroid hormone 1-34 (PTH) (Bachem California, PCAL100)
is prepared in vehicle (10.sup.-3 N HCl, 0.15 M NaCl, 20 mg/ml
cysteine-HCl) at the following concentrations: 0.156, 0.47, 1.56,
4.7, 15.6, and 156 .mu.M. Alzet mini-osmotic pumps are filled with
the PTH solution and maintained in 37.degree. C. saline for 4 hours
prior to implantation.
Serum Samples
Rats are anesthetized by CO.sub.2 from dry ice and daily blood
samples are obtained via cardiac puncture using a 27 gauge needle.
Baseline samples are taken just prior to TPTX. Daily samples are
obtained in the morning. Samples are allowed to clot on their side
for several hours and subsequently spun at 1000.times.g for 15
minutes to obtain serum. Serum is aliquoted and stored in the
refrigerator until assayed for serum calcium. Serum calcium is
measured (see above) daily for at least 7 days following TPTX.
Uses of Compounds of This Invention
Compounds of this invention which bind to an SH2 domain of interest
may be used as biological reagents in assays as described herein
for functional classification of a pTyr-binding domain (e.g. SH2 or
Pi domain) of a particular protein, particularly a newly discovered
protein. Families or classes of such proteins which bind to
pTyr-containing ligands may now be defined functionally, with
respect to ligand specificity. Moreover, compounds of this
invention can be used to inhibit the occurrence of biological
events resulting from molecular interactions mediated by the
protein of interest. Inhibiting such interactions can be useful in
research aimed at better understanding the biology of events
mediated by the binding of pTyr-containing ligands to their
receptors.
Such compounds would be useful, for example, in the diagnosis,
prevention or treatment of conditions or diseases resulting from a
cellular processes mediated by the binding of a pTyr-containing
ligand with a receptor therefor. For example, a patient can be
treated to prevent the occurrence or progression of osteoporosis or
to reverse its course by administering to the patient in need
thereof an SH2inhibitor which selectively binds Src SH2 or
otherwise interferes with Src-mediated signaling.
There are many other conditions for which such signal transduction
inhibitors may be useful therapeutically, including, e.g., breast
cancer where the SH2 domain-containing proteins Src, PLCgamma and
Grb7 have been implicated. Other relevant conditions include
prostate cancer, in which case targeting Grb2, PLCgamma, and P13K,
all of which contain SH2 domains, may be useful in treatment or
prevention of the disease. Inhibition of the interaction of Grb2 or
Abl SH2 domains with BCR-abl may be useful to treat chronic
myelogenous leukemia (CML) or acute myelogenous leukemia (AML).
Still other relevant applications include the prevention of
interferon-, growth factor-, or cytokine-mediated diseases (e.g.
inflammatory diseases) by targeting the interaction of STAT
proteins with their pTyr-containing ligands or otherwise inhibiting
their signal transduction pathways. Agents that block the SH2
domains of ZAP-70 or otherwise inhibit ZAP-70-mediated signaling
would be candidates for the treatment of immune-related disorders
such as rejection of transplanted bone marrow, skin or other
organs; rheumatoid arthritis; inflammatory bowel disease; and
systemic lupus erythmatosis, and a variety of autoimmune
diseases.
By virtue of the capacity to inhibit protein--protein interactions
or a relevant kinase or phosphatase activity required for cellular
events of pharmacologic importance, compounds of this invention
which inhibit cellular signal transduction may be used in
pharmaceutical compositions and methods for treatment or prevention
in a subject in need thereof. Such inhibitors can be used to treat
or reduce the risk of the diseases or their pathological effects
mediated by such interactions.
For example, drugs that completely block one of the two ZAP SH2
domains should effectively prevent ZAP from associating with the
activated TCR and thus block T cell activation. A ZAP antagonist or
inhibitor would specifically inhibit T cells and avoid the toxicity
of the currently used immunosuppressive drugs, FK506 and
cyclosporin, which target the more ubiquitously expressed protein,
calcineurin. Since calcineurin is required for cellular activities
in several tissues in addition to T cells, cyclosporin and FK506
cause side effects in the kidney and central nervous system which
limit their application largely to patients with organ transplant
rejection.
Therapeutic/Prophylactic Administration & Pharmaceutical
Compositions
Compounds of this invention can exist in free form or, where
appropriate, in salt form. Pharmaceutically acceptable salts of
many types of compounds and their preparation are well-known to
those of skill in the art. The pharmaceutically acceptable salts of
compounds of this invention include the conventional non-toxic
salts or the quatemary ammonium salts of such compounds which are
formed, for example, from inorganic or organic acids of bases.
The compounds of the invention may form hydrates or solvates. It is
known to those of skill in the art that charged compounds form
hydrated species when lyophilized with water, or form solvated
species when concentrated in a solution with an appropriate organic
solvent.
This invention also relates to pharmaceutical compositions
comprising a therapeutically (or prophylactically) effective amount
of the compound, and a pharmaceutically acceptable carrier or
excipient. Carriers include e.g. saline, buffered saline, dextrose,
water, glycerol, ethanol, and combinations thereof, and are
discussed in greater detail below. The composition, if desired, can
also contain minor amounts of wetting or emulsifying agents, or pH
buffering agents. The composition can be a liquid solution,
suspension, emulsion, tablet, pill, capsule, sustained release
formulation, or powder. The composition can be formulated as a
suppository, with traditional binders and carriers such as
triglycerides. Oral formulation can include standard carriers such
as pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
Formulation may involve mixing, granulating and compressing or
dissolving the ingredients as appropriate to the desired
preparation.
The pharmaceutical carrier employed may be, for example, either a
solid or liquid.
Illustrative solid carrier include lactose, terra alba, sucrose,
talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic
acid and the like. A solid carrier can include one or more
substances which may also act as flavoring agents, lubricants,
solubilizers, suspending agents, fillers, glidants, compression
aids, binders or tablet-disintegrating agents; it can also be an
encapsulating material. In powders, the carrier is a finely divided
solid which is in admixture with the finely divided active
ingredient. In tablets, the active ingredient is mixed with a
carrier having the necessary compression properties in suitable
proportions ,and compacted in the shape and size desired. The
powders and tablets preferably contain up to 99% of the active
ingredient. Suitable solid carriers include, for example, calcium
phosphate, magnesium stearate, talc, sugars, lactose, dextrin,
starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl
cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange
resins.
Illustrative liquid carriers include syrup, peanut oil, olive oil,
water, etc. Liquid carriers are used in preparing solutions,
suspensions, emulsions, syrups, elixirs and pressurized
compositions. The active ingredient can be dissolved or suspended
in a pharmaceutically acceptable liquid carrier such as water, an
organic solvent, a mixture of both or pharmaceutically acceptable
oils or fats. The liquid carrier can contain other suitable
pharmaceutical additives such as solubilizers, emulsifiers,
buffers, preservatives, sweeteners, flavoring agents, suspending
agents, thickening agents, colors, viscosity regulators,
stabilizers or osmo-regulators. Suitable examples of liquid
carriers for oral and parenteral administration include water
(partially containing additives as above, e.g. cellulose
derivatives, preferably sodium carboxymethyl cellulose solution),
alcohols (including monohydric alcohols and polyhydric alcohols,
e.g. glycols) and their derivatives, and oils (e.g. fractionated
coconut oil and arachis oil). For parenteral administration, the
carrier can also be an oily ester such as ethyl oleate and
isopropyl myristate. Sterile liquid carders are useful in sterile
liquid form compositions for parenteral administration. The liquid
carrier for pressurized compositions can be halogenated hydrocarbon
or other pharmaceutically acceptable propellant. Liquid
pharmaceutical compositions which are sterile solutions or
suspensions can be utilized by, for example, intramuscular,
intraperitoneal or subcutaneous injection. Sterile solutions can
also be administered intravenously. The compound can also be
administered orally either in liquid or solid composition form.
The carrier or excipient may include time delay material well known
to the art, such as glyceryl monostearate or glyceryl distearate
along or with a wax, ethylcellulose, hydroxypropylmethylcellulose,
methylmethacrylate and the like. When formulated for oral
administration, 0.01% Tween 80 in PHOSAL PG-50 (phospholipid
concentrate with 1,2-propylene glycol, A. Nattermann & Cie.
GmbH) has been recognized as providing an acceptable oral
formulation for other compounds, and may be adapted to formulations
for various compounds of this invention.
A wide variety of pharmaceutical forms can be employed. If a solid
carrier is used, the preparation can be tableted, placed in a hard
gelatin capsule in powder or pellet form or in the form of a troche
or lozenge. The amount of solid carrier will vary widely but
preferably will be from about 25 mg to about 1 g. If a liquid
carrier is used, the preparation will be in the form of a syrup,
emulsion, soft gelatin capsule, sterile injectable solution or
suspension in an ampule or vial or nonaqueous liquid
suspension.
To obtain a stable water soluble dosage form, a pharmaceutically
acceptable salt of the compound may be dissolved in an aqueous
solution of an organic or inorganic acid, such as a 0.3M solution
of succinic acid or citric acid. Alternatively, acidic derivatives
can be dissolved in suitable basic solutions. If a soluble salt
form is not available, the compound is dissolved in a suitable
cosolvent or combinations thereof. Examples of such suitable
cosolvents include, but are not limited to, alcohol, propylene
glycol, polyethylene glycol 300, polysorbate 80, glycerin,
polyoxyethylated fatty acids, fatty alcohols or glycerin hydroxy
fatty acids esters and the like in concentrations ranging from
0-60% of the total volume.
Various delivery systems are known and can be used to administer
the compound, or the various formulations thereof, including
tablets, capsules, injectable solutions, encapsulation in
liposomes, microparticies, microcapsules, etc. Methods of
introduction include but are not limited to dermal, intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, pulmonary, epidural, ocular and (as is usually
preferred) oral routes. The compound may be administered by any
convenient or otherwise appropriate route, for example by infusion
or bolus injection, by absorption through epithelial or
mucocutaneous linings (e.g., oral mucosa, rectal and intestinal
mucosa, etc.) and may be administered together with other
biologically active agents. Administration can be systemic or
local. For treatment or prophylaxis of nasal, bronchial or
pulmonary conditions, preferred routes of administration are oral,
nasal or via a bronchial aerosol or nebulizer.
In certain embodiments, it may be desirable to administer the
compound locally to an area in need of treatment; this may be
achieved by, for example, and not by way of limitation, local
infusion during surgery, topical application, by injection, by
means of a catheter, by means of a suppository, or by means of a
skin patch or implant, said implant being of a porous, non-porous,
or gelatinous material, including membranes, such as sialastic
membranes, or fibers.
In a specific embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic to
ease pain at the side of the injection. Generally, the ingredients
are supplied either separately or mixed together in unit dosage
form, for example, as a lyophilized powder or water free
concentrate in a hermetically sealed container such as an ampoule
or sachette indicating the quantity of active agent. Where the
composition is to be administered by infusion, it can be dispensed
with an infusion bottle containing sterile pharmaceutical grade
water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
Administration to an individual of an effective amount of the
compound can also be accomplished topically by administering the
compound(s) directly to the affected area of the skin of the
individual. For this purpose, the compound is administered or
applied in a composition including a pharmacologically acceptable
topical carrier, such as a gel, an ointment, a lotion, or a cream,
which includes, without limitation, such carriers as water,
glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides,
fatty acid esters, or mineral oils.
Other topical carriers include liquid petroleum, isopropyl
palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene
monolaurate (5%) in water, or sodium lauryl sulfate (5%) in water.
Other materials such as anti-oxidants, humectants, viscosity
stabilizers, and similar agents may be added as necessary.
Percutaneous penetration enhancers such as Azone may also be
included.
In addition, in certain instances, it is expected that the compound
may be disposed within devices placed upon, in, or under the skin.
Such devices include patches, implants, and injections which
release the compound into the skin, by either passive or active
release mechanisms.
Materials and methods for producing the various formulations are
well known in the art and may be adapted for practicing the subject
invention. See e.g. U.S. Pat. Nos. 5,182,293 and 4,837,311
(tablets, capsules and other oral formulations as well as
intravenous formulations) and European Patent Application
Publication Nos. 0 649 659 (published Apr. 26, 1995; illustrative
formulation for IV administration) and 0 648 494 (published Apr.
19, 1995; illustrative formulation for oral administration).
The effective dose of the compound will typically be in the range
of about 0.01 to about 50 mg/kgs, preferably about 0.1 to about 10
mg/kg of mammalian body weight, administered in single or multiple
doses. Generally, the compound may be administered to patients in
need of such treatment in a daily dose range of about 1 to about
2000 mg per patient.
The amount of compound which will be effective in the treatment or
prevention of a particular disorder or condition will depend in
part on the nature and severity of the disorder or condition, which
can be determined by standard clinical techniques. In addition, in
vitro or in vivo assays may optionally be employed to help identify
optimal dosage ranges. Effective doses may be extrapolated from
dose-response curves derived from in vitro or animal model test
systems. The precise dosage level should be determined by the
attending physician or other health care provider and will depend
upon well known factors, including route of administration, and the
age, body weight, sex and general health of the individual; the
nature, severity and clinical stage of the disease; the use (or
not) of concomitant therapies.
The invention also provides a pharmaceutical pack or kit comprising
one or more containers filled with one or more of the ingredients
of the pharmaceutical compositions of the invention. Optionally
associated with such container(s) can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceutical products, which notice reflects approval
by the agency of manufacture, use or sale for human
administration.
The representative examples which follow are intended to help
illustrate the invention, and are not intended to, nor should they
be construed to, limit the scope of the invention. Indeed, various
modifications of the invention and many further embodiments
thereof, in addition to those shown and described herein, will
become apparent to those skilled in the art from the full contents
of this document, including the examples which follow and the
references to the scientific and patent literature cited herein. It
should further be appreciated that the contents of those cited
references are incorporated herein by reference to help illustrate
the state of the art.
In addition, the full contents of U.S. patent applications U.S.
Ser. Nos. 08/968,490 and 09/190,424(Weigele et al, "Novel Signal
Transduction Inhibitors, Compositions Containing Them & Uses
Thereof" (filed Nov. 12, 1198 and Nov. 12, 1999, respectively) and
WO 99/24442, as well as U.S. Ser. Nos. 60/078,412 and 60/108,084
(Buchanan et al, "Novel Signal Transduction Inhibitors,
Compositions Containing Them & Uses Thereof", filed Mar. 18,
1998 and Nov. 12, 1998, respectively) and WO 99/47529 are
incorporated by reference herein. Those documents provide
additional synthetic and other guidance which may be of interest to
the practitioner of the subject invention.
The following examples contain important additional information,
exemplification and guidance which can be adapted to the practice
of this invention in its various embodiments and the equivalents
thereof.
EXAMPLES
Example 1
{4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7.
8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenoxy}-acet
ic acid ##STR20##
(a) 2-Cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene
To a mixture of 6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ol
(Helvetica Chimica Acta 1947, 1883.) (9.9 g, 61.0 mmol) and
Cs.sub.2 CO.sub.3 (26.3 g, 73.2 mmol) in DMF (100 mL) was added
(bromomethyl)cycycloxane (10.2 mL, 73.2 mmol). The mixture was
heated to 60.degree. C. for 18 h, dumped into water and extracted
with ethyl acetate. The combined extracts were washed with water,
dried over magnesium sulfate and concentrated to a solid. The solid
was recrystallized from ethanol (13.0 g, 83%). m.p. 62-63.degree.
C.
(b)
2-Cycycloxylmethoxy-6,7,8,9-tetrahydro-benzocyclohepten-5-one
A mixture of
2-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene (0.50
g, 1.94 mmol), potassium persulfate (1.57 g, 5.82 mmol), copper(II)
sulfate pentahydrate (0.48 g, 1.94 mmol) and CH.sub.3 CN/H.sub.2 0
(1:1, 13 mL) were heated at reflux for 45 min. After cooling the
mixture was diluted with water and extracted with EtOAc. The
combined extracts were washed with water, dried over magnesium
sulfate, concentrated, and chomatographed over silica gel (20%
EtOAc/hexane) to a white solid (0.46 g, 87%). MS [M +H] 273, m.p.
86-88.degree. C.
(c)
2-Cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol
To a suspension of
2-cycycloxylmethoxy-6,7,8,9-tetrahydro-benzocyclohepten-5-one (14.5
g, 53.23 mmol) in EtOH (150 mL) was added NaBH.sub.4 (1.96 g, 53.23
mmol) portionwise over 10 min. The mixture was heated to 40 OC for
1 h, cooled to 0.degree. C., then made acidic with the addition of
1 N HCl. The white precipated solids were filtered, washed with
water, and dried in vacuuo (14.25 g, 97%). m.p. 102-103.degree.
C.
(d)
5-Azido-2-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene
To 2-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol
(14.2 g, 52.0 mmol) in toluene (100 mL) at 0.degree. C. was added
DBU (9.4 mL, 62.9 mmol) followed by diphenylphosphoryl azide (13.6
mL, 62.9 mmol). The mixture was allowed to stir for 18 h at rt,
diluted with EtOAc, then washed successively with 1 N HCl, and
water. The organic layer was dried over MgSO.sub.4, filtered,
concentrated, and chomatographed over silica gel (5% EtOAc/hexane)
to yield a 7:3 mixture of
5-azido-2-cycycloxylnethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene/
3-cycycloxylmethoxy-6,7-dihydro-5H-benzocycloheptene (12.6 g). This
mixture was used without further purification in the next
reaction.
(e)
(2-Cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-H)-carbamic
Acid tert-butyl Ester
To the mixture from the preceeding reaction dissolved in EtOAc (150
mL) was added 10% Pd/C (500 mg) followed by Boc.sub.2 O (5.61 g, 25
mmol). The mixture was hydrogenated at STP for 17 h, filtered
though glass, concentrated, and chomatographed over silica gel to a
white solid (7.93 g, 41% from (c)). MS [M-H].sup.- 372, m.p.
125-126.degree. C.
(f)
(3-Bromo-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-yl)-
carbamic Acid tert-butyl Ester
To
(2-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-yl)-carbamic
acid tert-butyl ester (7.93 g, 21.2 mmol) in CH.sub.3 CN (300 mL)
was added NBS (3.96 g, 22.3 mmol). The mixture was stirred for 1.5
h, then blown dry under a stream of N.sub.2. The residue was
resuspended in CCl.sub.4, filtered, and concentrated to a solid.
The solid was recrystallized from CH.sub.3 CN to yield a white
solid (8.5 g, 88%). MS [M-H].sup.- 452, m.p. 142-144.degree. C.
(g)
9-tert-Butoxycarbonylamino-3-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzo
cycloheptene-2-carboxylic Acid
To
(3-bromo-2-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-yl)-
carbamic acid tert-butyl ester (8.0 g, 17.7 mmol) in THF (100 mL)
at -78.degree. C. was added nBuLi (21.2 mL, 53.1 mmol, 2.5 M
hexane) dropwise. The mixture was stirred for 10 min then a stream
of CO.sub.2 (g) was bubbled though the mixture for 2 min. The
mixture was diluted with ether and water, warmed to rt and the
layers seperated. The aqueous layer was extracted with Et.sub.2 O
and the combined extracts were washed with water, dried over
magnesium sulfate, and concentrated to a solid. The solid was
recrystallized from Et.sub.2 O to yield a white solid (5.0 g, 68%).
MS [M-H].sup.- 416, m.p. 159-160.degree. C.
(h)
(3-Carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-
yl)-carbamic Acid tert-butyl Ester
To
9-tert-butoxycarbonylamino-3-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzo
cycloheptene-2-carboxylic acid (1.5 g, 3.60 mmol) in CH.sub.2
Cl.sub.2 /DMF (4:1, 25 mL) was added HOBT (0.53 g, 4.0 mmol) and
EDC (0.76 g, 4.0 mmol) and the mixture allowed to stir for 30 min.
To this was added NH.sub.40 H (27% aqueous, 0.30 mL, 4.0 mmol) and
stirring was continued for 3 h. The mixture was diluted with 1 M
HCl and extracted with EtOAc. The combined extracts were washed
with water, sat'd NaHCO.sub.3, dried over magnesium sulfate, and
concentrated to a solid. The solid was recrystallized from Et.sub.2
O to yield a white solid (5.0 g, 68%). MS [M-H].sup.+ 415, m.p.
193-194.degree. C.
(i)
9-Amino-3-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carb
oxylic Acid Amide
To
(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-
yl)-carbamic acid tert-butyl ester (0.60 g, 1.44 mmol) in CH.sub.2
C1.sub.2 (8 mL) was added TFA (2 mL). The mixture was stirred for 1
h, evaporated under a stream of N.sub.2, diluted with CH.sub.2
Cl.sub.2 and washed with 1 N NaOH. The organic layer was dried over
magnesium sulfate and concentrated to a tan solid (0.45 g, 99%). MS
[M+H].sup.+ 316, m.p. 170-172.degree. C.
(j)
(R,S)-9-[(S)-2-Acetylamino-3-(4-hydroxy-phenyl-propionylamino]-3-cycycloxy
lmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carboxylic Acid
Amide
To N-acetyl-L-tyrosine (0.12 g, 0.55 mmol) in 5 mL of DME at
0.degree. C. was added HOBT (0.083 g, 0.55 mmol), EDC (0.10 g, 0.55
mmol) and
9-amino-3-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carb
oxylic acid amide (0.16 g, 0.50 mmol). The resulting solution was
allowed to stir for 3 h. The clear, yellow solution was diluted
with 75 mL of ethyl acetate and was washed with water, 1N HCl,
saturated NaHCO.sub.3, saturated NH.sub.4 Cl and brine. Drying over
MgSO.sub.4 and concentration yielded a white foamy solid (0.24 g,
92%). MS [M+H].sup.+ 521.
(k)
{4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydr
o-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-phenoxy}-acetic
Acid tert-butyl Ester
To
(R,S)-9-[(S)-2-acetylamino-3-(4-hydroxy-phenyl)-propionylamino]-3-cycyclox
ylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carboxylic acid
amide
(0.10 g, 0.19 mmol) in DMF (2 mL) was added potassium carbonate
(8.0 mg, 0.58 mmol) and tert-butyl bromoacetate (85 [L, 0.58 mmol).
The suspension was stirred at room temperature under an atmosphere
of N.sub.2 for 48 hours, after which the solution was diluted with
25 mL ethyl acetate and washed with water, 1N HCl, and brine.
Drying over MgSO.sub.4 and concentration yielded the crude ester
which was used without purification immediately in the next
reaction.
(l)
{4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohecylmethoxy-6,7,8,9-tetrahydr
o-5H-benzocyclohepten-(R or S)-5-ylcarbamoyl-ethyl]-phenoxy}-acetic
Acid
To
{4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydr
o-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-phenoxy}-acetic
acid tert-butyl ester (crude from the preceeding reaction) in
methylene chloride (2 mL) was added TFA (0.25 mL). The mixture was
stirred for 20 min., evaporated to dryness, and dissolved in DMSO
(2 mL). Purification by RP HPLC (CH.sub.3 CN/H.sub.2 O) and
lyophylization yielded two isomers: Isomer 1 (S,S): white solid
(9.6 mg, 8.5%). MS M-H].sup.+ 580. Isomer 2 (S,R): white solid (34
mg, 7.7%). MS M-H].sup.+ 580.
Example 2
Phosphoric acid
mono-{4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cycloxlmethoxy-6,7,8,9-tetrahy
dro-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-phenyl} Ester
##STR21##
(a) Phosphoric acid
4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro
-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-phenyl Ester
Dibenzyl Ester
To
(S)-9-[(S)-2-acetylamino-3-(4-hydroxy-phenyl)-propionylamino]-3-cycycloxyl
methoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carboxylic acid
amide (0.13 g, 0.24 mmol) in CH.sub.3 CN at -20 OC were added in
succession DIEA (0.10 mL, 0.57 mmol), CCl.sub.4 (0.15 mL, 1.55
mmol), DMAP (3.7 mg, 0.030 mmol), and dibenzylphosphite (0.10 mL,
0.45 mmol). The mixture was stirred for 3 h, diluted with water,
and extracted with EtOAc. The combined extracts were washed with
water, 1 N HCl, sat'd NaHCO.sub.3, dried over NaSO.sub.4,
concentrated, and chomatographed over silica gel (5% MeOH/CH.sub.2
Cl.sub.2) to give a semi-solid (0.12 g, 65%). MS [M+H.sup.+
782.
(b) Phosphoric acid
mono-{4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetr
ahydro-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-phenyl}
Ester
To phosphoric acid
4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro
-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-phenyl ester
dibenzyl ester (0.12 g, 0.16 mmol) in EtOH (5 mL) was added 20%
Pd/C (100 mg). The mixture was hydrogenated at STP for 2 h. The
mixture was filtered though glass, concentrated and purified by RP
HPLC (CH.sub.3 CN/H.sub.2 O). Lyophylization yielded a white solid
(70 mg, 97%). MS M-H].sup.+ 602.
Example 3
({4-[(S)-2-Acetylamino-2(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro
-5H-benzocyclohepten-(R or S)-5-ylcarbamoyl-ethyl]-phenyl)
-difluoro-methyl)-phosphonic Acid ##STR22##
(a)
({4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahyd
ro-5H-benzocyclohepten-(L or
S)-5-ylcarbamoyl)-ethyl-phenyl}-difluoro-methyl)-phosphonic acid
diethyl ester
To
(S)-2-acetylamino-3-{4-[(diethoxy-phosphoryl)-difluoro-methyl]-phenyl}-pro
pionic acid (Tetrahedron Lett. 1993, 34, 4125) (0.14 g, 0.38 mmol)
in CH.sub.2 Cl.sub.2 /DMF (4:1, 4 mL) at 0.degree. C. was added
HOBT (0.063 g, 0.46 mmol) and EDC (0.085 g, 0.44 mmol). To this was
added
9-amino-3-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carb
oxylic acid amide (0.11 g, 0.38 mmol) and stirring was continued
for 1 h. The mixture was then diluted with EtOAc (70 mL), washed
with 1 N HCl, sat'd NaHCO.sub.3, sat'd NH.sub.4 Cl, and brine. The
organic layer was dried over MgSO.sub.4, filtered, and concentrated
to a foamy solid (0.19 g, 84%).
Purification by RP HPLC (CH.sub.3 CN/H.sub.2 O) and lyophylization
yielded two isomers:
Isomer 1: white solid (52 mg, 20%). MS [M-H].sup.+ 668.
Isomer 2: white solid (90 mg, 36%). MS M-H].sup.+ 668.
(b)
({4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahyd
ro-5H-benzocyclohepten-(S)-5-ylcarbamoyl-ethyl]-phenyl}-difluoro-methyl)-ph
osphonic Acid
To Isomer 1 (preceeding reaction) (53 mg, 0.079 mmol) in CH.sub.3
CN (3 mL) cooled to -20.degree. C. was added TMSI (0.15 mL, 1.0
mmol). The mixture was stirred for 20 min, quenched with sat'd
NaHCO.sub.3 (5 mL), and purified by RP HPLC (CH.sub.3 CN/H.sub.2 O)
to a white solid (39.4 mg, 78%). MS [M-H].sup.+ 636.
(c)
({4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahyd
ro-5H-benzocyclohepten-(R)-5-ylcarbamoyl)-ethyl]-phenyl}-difluoro-methyl)-p
hosphonic Acid
Was made as for Isomer 1 (example 3, (b)). (62.3 mg, 70%). MS
[M-H].sup.+ 636.
Example 4
({4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydr
o-5H-benzocyclohepten-(S and
R)-5-ylcarbamoyl)-ethyl]-phenyl}-phosphono-methyl)-phosphonic Acid
##STR23##
(a) p(CH.sub.2 PO.sub.3 Et.sub.2)-L-Phe-OH
Fmoc-p(CH.sub.2 PO.sub.3 Et.sub.2)-L-Phe-OH (5.0 g, 9.3 mmol) was
dissolved in 170 mL of THF and 50 mL of diethyl amine and the
mixture was vigorously stirred at rt for 3 h. Solvents were removed
under reduced pressure and the solid was resuspended in anhydrous
ether, filtered, and dried on high vacuum to afford 2.8 g (94%) of
p(CH.sub.2 PO.sub.3 Et.sub.2)-L-Phe-OH as white solid which was
used without purification in the next step.
(b) N-Boc-p(CH.sub.2 PO.sub.3 Et.sub.2)-L-Phe-OH
To a solution of p(CH.sub.2 PO.sub.3 Et.sub.2)-L-Phe-OH (5.0 g,
16.7 mmol) in a 1:1 mixture of DME/water (140 ml) at 0.degree. C.
was added NaHCO.sub.3 (3.1 g, 36.8 mmol) followed by Boc.sub.2 O
(4.0 g, 18.4 mmol). The mixture was stirred at 0.degree. C. for 30
min then warmed to rt and stirred for 1 h. About 50 ml of DME was
removed by evaporation then the remaining aqueous solution was
extracted with EtOAc (2.times.50 mL). The aqueous layer was brought
to pH 4 with 1 N HCl and extracted with EtOAc (3.times.100 mL). The
combined extracts (second) were washed with water, dried over
MgSO.sub.4, filtered and concentrated to a colorless oil (6.2 g,
90%). MS [M-H].sup.- 414.
(c) N-Boc-p(CH.sub.2 PO.sub.3 Et.sub.2)-L-Phe-OMe
To a solution of N-Boc-p(CH.sub.2 PO.sub.3 Et.sub.2)-L-Phe-OH (5.1
g, 12.2 mmol) in DMF (60 mL) was added Cs.sub.2 CO.sub.3 (4.8 g,
14.7 mmol) followed by MeI (0.76 ml, 12.2 mmol). The mixture was
stirred for 1 h, diluted with water (600 ml) and extracted with
EtOAc (3.times.100 mL). The combined extracts were washed with
water, 10% NaHSO.sub.3, dried over MgSO.sub.4, filtered and
concentrated to a solid which was recrystallized from EtOAc/hexane
to give a white solid (4.6 g, 88%). MS [M-H].sup.- 428. m.p.
104-105.degree. C.
(d) N-Boc-p[CH(PO.sub.3 Et.sub.2).sub.2 ]-L-Phe-OMe
To a suspension of N-Boc-p(CH.sub.2 PO.sub.3 Et.sub.2)-L-Phe-OMe
(7.0 g, 16.3 mmol) in 185 mL of anhydrous DME, purged with N.sub.2
and cooled to -42.degree. C. (CH.sub.3 CN/dry ice), was added
dropwise lithium bis(trimethylsilyl)amide (1 M THF, 48.9 mL, 48,9
mmol). The reaction mixture was stirred at -42.degree. C. for 15
min. Diethylchlorophosphate (4.7 mL, 32.6 mmol) was added and the
orange solution was stirred at -42.degree. C. for an additional 20
min before being quenched with 1 N HCl (20 ml). The mixture was
further diluted with water and extracted with EtOAc (3.times.100
mL). The combined extracts were washed with water, dried over
MgSO.sub.4, filtered, concentrated, and chomatographed over silica
gel (3% MeOH/CH.sub.2 Cl.sub.2) to give a colorless oil (6.0 g,
65%). MS [M-H].sup.- 564.
(e) N-Boc-p[CH(PO.sub.3 Et.sub.2).sub.2 ]-L-Phe-OH
To a solution of N-Boc-p[CH(PO.sub.3 Et.sub.2).sub.2 ]-L-Phe-OMe
(0.49 g, 0.966 mmol) in 5 mL of THF cooled to 0.degree. C. was
added dropwise a solution of lithium hydroxide monohydrate (49.0
mg, 1.17 mmol) in 1.0 mL of water. The reaction mixture was stirred
at 0.degree. C. for 1 h. THF was removed under reduced pressure to
a yellow oil which was diluted with 10 mL of 1 N HCl. The aqueous
phase was extracted with CH.sub.2 Cl.sub.2 (8.times.15 mL), and the
extracts were combined, dried over Na.sub.2 SO.sub.4, and
concentrated to afford 0.45 g (95%) of N-Boc-p[CH(PO.sub.3
Et.sub.2).sub.2 ]-L-Phe-OH as a crystalline white solid. MS
[M-H].sup.- 550. m.p. 84-87.degree. C.
(f)
[{4-[(S)-2-tert-Butoxycarbonylamino-2-(3-carbamoyl-2cycycloxylmethoxy-6,7,
8,9-tetrahydro-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-phenyl}-(die
thoxy-phosphoryl)-methyl-phosphonic acid diethyl ester
To N-Boc-p[CH(PO.sub.3 Et.sub.2).sub.2 ]-L-Phe-OH (0.22 g, 0.41
mmol) in CH.sub.2 C1.sub.2 /DMF (4:1, 4 mL) at 0.degree. C. was
added HOBT (0.066 g, 0.49 mmol) and EDC (0.092 g, 0.48 mmol). To
this was added
9-amino-3-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carb
oxylic acid amide (0.12 g, 0.38 mmol) and stirring was continued
for 1 h. The mixture was then diluted with EtOAc (70 mL), washed
with 1 N HCl, sat'd NaHCO.sub.3, sat'd NH.sub.4 Cl, and brine. The
organic layer was dried over MgSO.sub.4, filtered, and concentrated
to a foamy solid (0.29 g, 90%). This material was used without
further purification in the next step.
(g)
[{4-[(S)-2-Amino-2-(3-carbam{yl-2-{cycloxylmethoxy-6,7,8,9-tetrahydro-5H-b
enzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-phenyl)-(diethoxy-phosphoryl-me
thyl]-phosphonic acid diethyl ester
To the crude material from the preceeding reaction (0.29 g, 0.34
mmol) in CH.sub.2 Cl.sub.2 (10 mL) was added TFA (2 mL). The
mixture was stirred for 30 min, evaporated to dryness and purified
by RP HPLC (CH.sub.3 CN/H.sub.2 O): Isomer 1: white solid (143 mg,
50%). MS M-H].sup.+ 750. Isomer 2: white solid (88 mg, 31%). MS
[M-H).sup.+ 750.
(h) f{4-[(S)-2-Acetylamino-2-(3-carbamoyl-2:
cycycloxylmethoxy-6,7,8,9tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)
-ethyl]-phenyl}-(diethoxy-phosphoryl-methyl]-phosphonic Acid
Diethyl Ester
To Isomer 1 (from the preceeding reaction) (0.14 g, 0.19 mmol) in
CH.sub.2 Cl.sub.2 (2 mL) at 0.degree. C. was added TEA (0.30 mL,
2.15 mmol) followed by Ac.sub.2 O (0.075 mL, 0.80 mmol). The
mixture was stirred for 30 min, diluted with CH.sub.2 Cl.sub.2, and
washed with 1 N HCl, sat'd NaHCO.sub.3, and sat'd NH.sub.4 Cl. The
organic layer was dried over MgSO.sub.4, filtered and concentrated
to a glassy solid (79.2 mg, 53%/o). This material (which was
homogeneous by HPLC) was used without purification in the next
reaction.
(i)
({4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahyd
ro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-phosphono-methyl)-
phosphonic Acid
To
[(4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahyd
ro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-(diethoxy-phosphor
yl)-methyl]-phosphonic acid diethyl ester (0.079 g, 0.10 mmol) in
CH.sub.3 CN at 0.degree. C. was added TMSI. The mixture was stirred
for 30 min, quenched with sat'd NaHCO.sub.3 (5 mL) and purified by
RP HPLC (CH.sub.3 CN/H.sub.2 O) to a white solid (61.2 mg, 90%). MS
[M-H].sup.+ 680.
(j)
((4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahyd
ro-5H-benzocyclohepten-(R)-5-ylcarbamoyl)-ethyl]-phenyl}-phosphono-methyl)-
phosphonic Acid
Was made as for Isomer 1 (example 4, (steps h and i)). (62.3 mg,
70%). MS [M-H].sup.+ 680.
Example 5
4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-
5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-formyl-benzoic acid
##STR24##
(a) 3-(1,3-Dithiolan-2-yl)-N-acetyl-L-tyrosine methyl ester
To L-3-formyl--N-acetyltyrosine methyl ester (U.S. Pat. No.
4,022,910) (0.13 g, 0.48 mmol) in methylene chloride (5 mL) at
0.degree. C. was added boron trifluoride diethyl etherate (0.12 mL,
0.96 mmol) followed by ethanedithiol (0.044 mL, 0.53 mmol). The
mixture was allowed to warm to rt and stirred for 1 h. The solution
was dumped into water and the layers separated. The aqueous layer
was extracted with methylene chloride and the combined extracts
were washed with water, dried over magnesium sulfate and
concentrated to a solid. The solid was recrystallized from ethyl
acetate/hexane (0.14 g, 82%). m.p. 97-99.degree. C.
(b)
3-(1,3-Dithiolan-2-yl)-4-(trifluoromethansulfonyloxy)-N-acetyl-L-phenylala
nine Methyl Ester
To 3-(1,3-dithiolan-2-yl)-N-acetyltyrosine methyl ester (0.50 g,
1.46 mmol) and triethylamine (0.22 mL, 1.61 mmol) in methylene
chloride (10 mL) at 0.degree. C. was added
N-phenyltrifluoromethanesulfonimide (0.58 g, 1.61 mmol). The
mixture was allowed to stir for five days then washed sequentially
with 1 N NaOH, 1 N HCl, and brine. The organic layer was dried over
magnesium sulfate and concentrated to a solid. The solid was
recrystallized from ethyl acetate/hexane (0.61 g, 87%). m.p.
116-118.degree. C.
(c)
3-(1.3-Dithiolan-2-yl)4-(carboxymethyl)-N-acetyl-L-phenylalanine
Methyl Ester
To
3-(1,3-dithiolan-2-yl)4-(trifluoromethansulfonyloxy)-N-acetyl-L-phenylalan
ine methyl ester (0.51 g, 1.08 mmol) in DMSO/MeOH (3:2, 5 mL) was
added triethylamine (0.33 mL, 2.36 mmol) followed by palladium
acetate (0.0073 g, 0.033 mmol) and
1,3-bis(diphenylphosphino)propane (0.013 g, 0.034 mmol). Carbon
monoxide was bubbled though for 3 min and the mixture was heated to
80.degree. C. for 24 hours. After cooling, the solution was diluted
with water and extracted with ethyl acetate. The combined extracts
were washed with water, dried over magnesium sulfate and
concentrated to a solid. The solid was recrystallized from ethyl
acetate/hexane (0.30 g, 72%). m.p. 113-119.degree. C.
(d)
3-(1.3-Dithiolan-2-yl)-4-(carboxymethyl)-N-acetyl-L-phenylalanine
To 3-(1,3-dithiolan-2-yl)4-(carboxymethyl)-N-acetyl-L-phenylalanine
methyl ester (0.22 g, 0.57 mmol) in THF (10 mL) at 0.degree. C. was
added lithium hydroxide monohydrate (0.025 g, 0.60 mmol, 1 mL
water). The mixture was stirred for 45 min., diluted with water,
made acidic with 1 N HCl, and extracted with ethyl acetate. The
combined extracts were washed with water, dried over magnesium
sulfate and concentrated to a glassy solid which was homogeneous by
RP HPLC (0.18 g, 86%). MS [M-H].sup.- 368.
(e)
4-[(S-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro-
5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl1-2-[1,3]dithiolan-2-yl-benzo
ic Acid Methyl Ester
To
3-(1,3-dithiolan-2-yl)-4-(carboxymethyl)-N-acetyl-L-phenylalanine
(0.13 g, 0.35 mmol) in CH.sub.2 Cl.sub.2 /DMF (5:1, 5 mL) at
0.degree. C. was added HOBT (0.051 g, 0.35 mmol) and EDC (0.072 g,
0.35 mmol). The mixture was stirred for 10 min then
(R,S)-9-amino-3-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-
2-carboxylic acid amide (0.10 g, 0.32 mmol) was added and stirring
was continued for 1 h. The solution was dumped into water and the
layers separated. The aqueous layer was extracted with methylene
chloride and the combined extracts were washed with water, 1N HCl,
dried over magnesium sulfate, and concentrated to a glassy solid
(0.20 g, 99%) which was homogeneous by RP HPLC. MS M-H].sup.-
636.
(f)
4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9tetrahydro-
5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-2-formyl-benzoic
acid methyl ester
To
4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro
-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-2-[1,3]dithiolan-2-yl-benz
oic acid methyl ester (0.20 g, 0.31 mmol) in CHCl.sub.3 /MeOH (1:1,
6 mL) was added mercury (II) perchlorate hydrate (0.38 g, 0.94
mmol). The mixture was stirred for 5 min then filtered though
Celite (CHCl.sub.3 wash). The organic layer was washed with 1 N
HCl, saturated NaHCO.sub.3, dried over magnesium sulfate, filtered,
and concentrated to a glassy solid (0.18 g) which was used
immediately in the next reaction.
(g)
4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro
-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-formyl-benzoic
Acid
To
4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro
-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-2-formyl-benzoic
acid methyl ester (0.18 g, 0.32 mmol) in THF (5 mL) was added
lithium hydroxide monohydrate (0.018 g, 0.42 mmol, 1 mL water). The
mixture was stirred for 1 h, acidified with TFA, and evaporated.
The residue was diluted with DMSO (2 mL) and purified by RP HPLC
(CH.sub.3 CN/H.sub.2 O). Lyophilization left a white solid (0.040
mg, 21%). MS [M-H].sup.- 576.
Example 6
{4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro
-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-phosphono-phenyl}-phosphon
ic Acid ##STR25##
(a) 2-tert-Butoxycarbonylamino-3-(3,4-dihydroxy-phenyl)-propionic
Acid Methyl Ester
To (3,4-(dihydroxyphenyl)-L-alanine methyl ester hydrochloride (5.4
g, 25.4 mmol) and Di-tert-butyl dicarbonate (5.5 g, 25.4 mmol) in a
mixture of THF (20 mL) and water (20 mL) at rt was added sodium
bicarbonate (3.2 g, 38.1 mmol). The mixture was allowed to stirred
for 16 h then washed with water, and extracted with EtOAc. The
organic layer was dried over magnesium sulfate, and concentrated to
a solid. The solid was recrystallized frome ethyl acetate/hexane (7
g, 88%). MS [M+H].sup.+ 312. m.p. 132-135.degree. C.
(b) 3-(3.
4-Bis-trifluoromethanesulfonyloxy-phenyl)-2-tert-butoxycarbonylamino-propi
onic Acid Methyl Ester
To 2-tert-Butoxycarbonylamino-3-(3,4-dihydroxy-phenyl)-propionic
acid methyl ester (12 g, 38.6 mmol) and triethyl amine (13 mL, 88.7
mmol) in methylene chloride (100 mL) at 0.degree. C. was added
N-phenyl-bis(trifluoromethanesulfonimide) (31.6 g, 88.7 mmol). The
mixture was allowed to stirred for two days then washed
sequentially with 1 N NaOH, 1 N HCl, and brine. The organic layer
was dried over magnesium sulfate, concentrated to a solid. The
solid was recrystallized from dichloremethane/hexane. MS
[M+Na].sup.+ 598. m.p. 80-82.degree. C.
(c)
3-[3,4-Bis-(diethoxy-phosphoryl)-phenyl]-2-tert-butoxycarbonylamino-propio
nic Acid Methyl Ester
To
3-(3,4-bis-trifluoromethanesulfonyloxy-phenyl)-2-tert-butoxycarbonylamino-
propionic acid methyl ester (2 g, 3.47 mmol), diethyl phosphite (1
mL, 7.65 mmol) and 4-methyl morpholine (0.93 mL, 8.3 mmol) in MeCN
(10 ml) was added Pd(PPh.sub.3).sub.4 (167 mg, 0.15 mmol). The
mixture was allowed to stirred for two days at 95.degree. C. then
diluted with saturated NH.sub.4 Cl and extracted with EtOAc. The
organic layer was dried over magnesium sulfate, concentrated, and
chomatographed over silica gel (5% MeOH/EtOAc) to an oil (0.2 g,
37% yield). MS [M+H].sup.+ 552 & [M+Na] 574.
(d) 3-[3.
4-Bis-(diethoxy-phosphoryl)-phenyl]-2-tert-butoxycarbonylamino-propionic
acid
To a solution of
3-[3,4-Bis-(diethoxy-phosphoryl)-phenyl]-2-tert-butoxycarbonylamino-propio
nic acid methyl ester (110 mg, 0.2 mmol) in 5 mL of THF cooled to
0.degree. C. was added dropwise a solution of lithium hydroxide
monohydrate (8.5 mg, 0.2 mmol) in 1.0 mL of water. The reaction
mixture was stirred at 0.degree. C. for 1 h. THF was removed under
reduced pressure to a yellow oil which was diluted with 10 mL of 1
N HCl. The aqueous phase was extracted with CH.sub.2 Cl.sub.2
(2.times.15 mL), and the extracts were combined, dried over
Na.sub.2 SO.sub.4, and concentrated to afford an oil 107 mg (100%).
MS [M-H].sup.- 537.
(f)
[4-[(S)-2-Amino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-b
enzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-(diethoxy-phosphoryl)-phenyl]-p
hosphonic Acid Diethyl Ester
To
3-(S)-[3,4-bis-(diethoxy-phosphoryl)-phenyl]-2-(2,2-dimethyl-propionylamin
o)-propionic acid (107 mg, 0.19 mmol) in a mixture of methylene
chloride/DMF (5:1, 10 mL) at 0.degree. C. was added HOBt (27 mg,
0.2 mmol) and EDC (27 mg, 0.2 mmol). The mixture was stirred for 10
min then (R,S)-9-amino-3-cycycloxylmethoxy-6,7,8,9-tetrahydro-5
H-benzocycloheptene-2-carboxylic acid amide (63 mg, 0.19 mmol) was
added and stirring was continued for 1 h. The solution was dumped
into water and the layers separated. The aqueous layer was
extracted with methylene chloride and the combined extracts were
washed with water, dried over magnesium sulfate and concentrated to
a glassy solid. This crude material was dissolved in methylene
chloride (10 mL) and 95% aqueous TFA (2 mL). The solution was
stirred for 1 h and then concentrated under a stream of N.sub.2.
The residue was purified by preparative RP HPLC. Elution with 40:60
MeCN-H.sub.2 0 (each containing 0.1% TFA) provided the 60 mg of the
title compound. MS [M+H].sup.+ 736.
(g)
[4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydr
o-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-(diethoxy-phosphoryl)-phe
nyl]-phosphonic Acid Diethyl Ester
To
[4-[(S)-2-amino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-b
enzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-(diethoxy-phosphoryl)-phenyl]-p
hosphonic acid diethyl ester (60 mg, 0.08 mmol) and triethyl amine
(0.1 mL, 0.8 mmol) in methylene chloride (10 mL) at 0.degree. C.
was added acetic anhydride (0.03 mL, 0.32 mmol). The mixture was
allowed to stirred for 1 h then diluted with water and extracted
with EtOAc. The combined extracts were dried over magnesium sulfate
and concentrated to a oil which was homogeneous by RP HPLC (65 mg,
crude).
(h)
(4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydr
o-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-phosphono-phenyl}-phospho
nic Acid
To a solution of
[4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydr
o-5-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-(diethoxy-phosphoryl)-phen
yl]-phosphonic acid diethyl ester (65 mg, 0.12 mmol) in MeCN (5 mL)
at -11.degree. C. was added TMSI (0.2 mL, 1.6 mmol). The mixture
was stirred for 3 h at -11.degree. C. and then quenched by
saturated NaHCO.sub.3 (1 mL). The resulting mixture was purified by
RP HPLC (CH.sub.3 CN/H.sub.2 O). Lyophilization left a white soild.
MS [M-H].sup.- 664.
Example 7
{4-[(S)-2-Amino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-be
nzocyclohepten-(R or
S)-5-ylcarbamoyl-ethyl]-2-phosphono-phenyl}-phosphonic Acid
##STR26##
The title compounds were made from intermediates in the synthesis
of example 6. MS [M-H].sup.- 622.
Example 8
({4-[(S)-2-Acetylamino-2-(7-carbamoyl-8-cyclohecylmethoxy-2,3,4,5-tetrahydr
o-benzo[b]oxepin-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-phosphono-methyl)-phosph
onic Acid ##STR27##
(a) 4-(4-Carbamoyl-3-hydroxy-phenoxy)-butyric Acid Ethyl Ester
To 2,4-dihydroxybenzamide (16.5 g, 108.3 mmol) in DMF (100 mL) was
added Cs.sub.2 CO.sub.3 (39.0 g, 119.1 mmol) followed by ethyl
4-bromobutyrate (17.0 mL, 119.1 mmol). The mixture was heated to 70
OC for 2 h, cooled, diluted with water and extracted with EtOAc.
The combined extracts were washed with water, dried over magnesium
sulfate, concentrated, and chomatographed over silica gel (60%
EtOAc/hexane) to a white solid (4.2 g, 15%). MS [M+H].sup.+
267.
(b) 4-(4-Carbamoyl-3:acycloxylmethoxy-phenoxy)-butyric Acid Ethyl
Ester
To 4-(4-carbamoyl-3-hydroxy-phenoxy)-butyric acid ethyl ester (3.0
g, 11.22 mmol) in DMF (25 mL) was added Cs.sub.2 CO.sub.3 (5.11 g,
15.71 mmol) followed by (bromomethyl)cycycloxane (1.72 mL, 12.34
mmol). The mixture was heated to 60.degree. C. for 6 h, then dumped
onto ice/H.sub.2 O. The formed solids were collected by filtration
(H.sub.2 O wash)and dried in vacuuo (3.74 g, 92%). MS [M+H].sup.+
364. m.p. 98-99.degree. C.
(c) 4-(4-Carbamoyl-3-cyclohexylmethoxy-phenoxy)-butyric Acid
To 4-(4-carbamoyl-3-cycycloxylmethoxy-phenoxy)-butyric acid ethyl
ester (3.74 g, 10.29 mmol) in THF (20 mL) was added LiOH-H.sub.2 O
(0.61 g, 14.4 mmol) and H.sub.2 O (6 mL). The mixture was stirred
for 30 min, concentrated, diluted with water, and acidified with 1
N HCl. White solids were filtered, washed with water, and dried in
vacuuo (3.37 g, 95%). MS [M+H].sup.+ 336.
(d)
8-Cycycloxylmethoxy-5-oxo-2,3,4,5-tetrahydro-benzo[b]oxepine-7-carboxylic
Acid Amide
4-(4-Carbamoyl-3-cycycloxylmethoxy-phenoxy)-butyric acid (3.4 g,
10.6 mmol) was added to PPA at 80 OC. The mixture was stirred for 1
h then dumped onto ice. The formed solids were filtered, washed
with water, dried, and further purified on silica gel (5%
MeOH/CH.sub.2 Cl.sub.2) to give an off-white solid (2.62 g, 82%).
MS [M+H].sup.+ 364. m.p. 185-186.degree. C.
(e)
8-Cycycloxylmethoxy-(R)-5-hydroxy-2,3,4,5-tetrahydro-benzo[b]oxepine-7-car
boxylic Acid Amide
To
8-cyclohexylmethoxy-5-oxo-2,3,4,5-tetrahydro-benzo[b]oxepine-7-carboxylic
acid amide (4.0 g, 13.3 mmol) suspended in CH.sub.2 Cl.sub.2 (25
mL) at -42.degree. C. was added (+)-DIP-Chloride (25 mL of CH.sub.2
Cl.sub.2). The mixture was stirred at -12.degree. C. for 18 h,
concentrated, diluted with Et.sub.2 O (200 mL) and treated with
diethanolamine (5.1 mL, 53.1 mmol). After stirring for 2.5 h, the
formed solids were filtered and washed with Et.sub.2 O. The
filtrate was concentrated, and the residue purified by silica gel
column chomatography (5% MeOH/CH.sub.2 C1.sub.2) to a white solid
(3.2 g, 80%). m.p. 164-165.degree. C. Optical purity determined by
Mosher Amide formation, 71% e.e.
(f)
(S)-5-Azido-8-cycycloxylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepine-7-carbo
xylic Acid Amide
This compound was prepared as for Example 1, step d.
(g)
(S)-5-Amino-8-cyclohexylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepine-7-carbo
xylic acid amide
To
(S)-5-azido-8-cycycloxylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepine-7-carbo
xylic acid amide (2.5 g, 5.8 mmol) in EtOAc (20 mL) was added 10%
Pd/C (0.25 g). The mixture was hydrogenated at STP for 17 h,
filtered though glass, and concentrated to a white solid (1.8 g,
98%). MS M-H].sup.- 317, m.p. 145-146.degree. C. Optical purity
determined by Mosher Amide formation, 55% e.e.
(h)
(14-[(S)-2-Acetylamino-2-(7-carbamoyl-8-cycycloxylmethoxy-2,3,4.
5-tetrahydro-benzo[b]oxepin-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-phosphono-me
thyl)-phosphonic Acid
This compound was prepared as for Example 4. MS [M-H].sup.-
680.
Example 9
({4-[2-(7-Carbamoyl-8-isobutoxy-2,3,4,5-tetrahydro-benzo[b]oxepin-(S)-5-ylc
arbamoyl)-(S)-2-(2,2-dimethyl-propionylamino)-ethyl]-phenyl}-phosphono-meth
yl)-phosphonic Acid ##STR28##
This compound was prepared as for Example 8. MS [M-H].sup.+
684.
Example 10
({4-[2-(7-Carbamoyl-8-isobutoxy-2,3,4,5-tetrahydro-benzo[b]oxepin-(R)-5-ylc
arbamoyl)-(S)-2-(2.
2-dimethyl-propionylamino)-ethyl]-phenyl}-phosphono-methyl)-phosphonic
acid ##STR29##
This compound was prepared as for Example 8. MS [M-H].sup.+
684.
Example 11
({4-[(S)-2-Amino-2-(7-carbamoyl-8-cycycloxylmethoxy-2,3,4,5-tetrahydro-benz
o[b]oxepin-(R or
S)-5-ylcarbamoyl)-ethyl]-phenyl}-phosphono-methyl-phosphonic Acid
##STR30##
The title compounds were made from intermediates in the synthesis
of example 8. MS [M-H].sup.- 638
Example 12
({4-[.sup.2
-(7-Carbamoyl-8-cycycloxylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepin-(R
or
S)-S-ylcarbamoyl)-(S)-2-phenylacetylamino-ethl]-phenyl}-phosphono-methyl-p
hosphonic Acid ##STR31##
These compound were prepared as for example 8. MS [M-H].sup.+
756.
Example 13
{4-[(S-2-Acetylamino-2-(7-carbamoyl-8-cycycloxylmethoxy-2,3,4,5-tetrahydro-
benzo[b]oxepin-(R or S)-5-ylcarbamoyl)-ethyl]-phenoxy}-acetic Acid
##STR32##
(R,S)-5-Amino-8-cycycloxylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepine-7-carb
oxylic acid amide was converted to the title compounds as for
example 1.
Example 14
{4-[2-(7-Carbamoyl-8-isobutoxy-2,3,4,5
tetrahydro-benzo[b]oxepin-(S)-5-ylcarbamoyl-(S)-2-(2,2-dimethyl-propioniyl
amino)-ethyl]-2-phoshono-phenyl}-phosphonic Acid ##STR33##
This compound was prepared using intermediates and procedures from
Example 6 and Example 8. MS [M-H].sup.+ 670.
Example 15
{4-[(S)-2-Amino-2-(7-carbamoyl-8-cycycloxylmethoxy-2,3,4,5-tetrahydro-benzo
[b]oxepin-(S)-5-ylcarbamoyl)-ethyl]-2-phosphono-phenyl}-phosphonic
Acid ##STR34##
This compound was prepared using intermediates and procedures from
Example 6 and Example 8. MS [M-H].sup.- 624.
Example 16
{4-[(S)-2-Acetylamino-2-(7-carbamoyl-8-cyclohexylmethoxy-2,3,4,5-tetahydro-
benzo[b]oxepin-(S)-5-ylcarbamoyl)-ethl]-2-phosphono-phenyl}-phosphonic
Acid ##STR35##
This compound was prepared using intermediates and procedures from
Example 6 and Example 8. MS [M-H].sup.- 664.
Example 17
{4-[2-(7-Carbamoyl-8-cyclohexylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepin-(S
)-5-ylcarbamoyl)-2-phenylacetylamino-ethyl]-2-phosphono-phenyl}-phosphonic
Acid ##STR36##
This compound was prepared using intermediates and procedures from
Example 6 and Example 8. MS [M-H].sup.- 742.
Example 18
{4-[(S)-2-Acetylamino-2-(7-carbamoyl-8-cycycloxylmethoxy-2,3,4,5-tetrahydro
-benzo[b]oxepin-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-phosphonic Acid
##STR37##
This compound was prepared using an intermediate from Example 8 and
from Bioorg. Med. Chem. Lett. 1997, 7, 1909. MS [M-H].sup.-
586.
Example 19
[(4-{(S)-2-Acetylamino-(S)-2-[1-(4-carbamoyl-7,8-dihydro-6H-5-oxa-9-thia-be
nzocyclohepten-2-yl)-ethylcarbamoyl]-ethyl}-phenyl)-difluoro-methyl]-phosph
onic Acid ##STR38##
(a) 2-(3-Hydroxy-propylsulfanyl)-phenol
2-Hydroxythiophenol (1.00 g, 8.70 mmol) was added to a mixture of
DMF (10 mL) and Cs.sub.2 CO.sub.3 (2.90 g, 8,90 mmol). To this was
added 3-bromopropanol (0.80 mL, 9.16 mmol) and the mixture was
stirred for 20 min. The mixture was added to into water and
extracted with EtOAc. The combined extracts were washed with water,
dried over magnesium sulfate and concentrated to a clear oil (2.36
g, 100%). MS [M-H].sup.- 183.
(b) 7,8-Dihydro-6H-5-oxa-9-thia-benzocycloheptene
To 2-(3-hydroxy-propylsulfanyl)-phenol (29.2 g, 158.5 mmol) in THF
(450 mL) was added triphenylphosphine (52.0 g, 200.0 mmol). The
solution was cooled to -40.degree. C. and diethyl azodicarboxylate
(31.5 mL, 164.0 mmol) was added slowly. The solution was warmed to
rt and stirred for 2.5 h. The THF was removed by evaporation and
the residue was treated with 1 L of Et.sub.2 O. The formed solids
were filtered off, and the filtrate concentrated to an oil which
was purified over silica gel (10% Et.sub.2 O/hexane) to a pink oil
(16.2 g, 61%).
(c) 7,8-Dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carboxylic
Acid
To 7,8-dihydro-6H-5-oxa-9-thia-benzocycloheptene (16.2 g, 97 mmol)
in 250 mL of dry hexane was added tetramethylethylene diamine (16
mL, 106 mmol). The solution was cooled to 0.degree. C. and
n-butyllitium (1.6 M solution in hexane, 73 mL, 116.8 mmol) was
slowly added with stirring. A tan-colored precipitate slowly
started to form and some gas evolution occured. The suspension was
stirred at rt for 18 h, after which CO.sub.2 gas was bubbled though
it for 20 min. An exothermic reaction occured. The mixture was
diluted with 300 mL ethyl acetate and 4 N HCl. After all solids
dissolved, the organic layer was washed with 1 N HCl. The acid was
extracted into the water layer using sat. NaHCO.sub.3. The aqueous
layer was washed with ethyl acetate and treated with 10 N HCl to pH
1. Extraction with ethyl acetate (2.times.250 mL), drying over
Na.sub.2 SO.sub.4 and concentration yielded the compound as a tan
solid (16.5 g, 81%). MS [M-H].sup.- 209.
(d) 7,8-Dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carboxylic Acid
Amide
To 7,8-dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carboxylic acid
(16.5g, 78.4 mmol) in 100 mL DMF was added in succession solid HOBT
(21.3g, 157.3 mmol), solid EDC hydrochloride (30.1 g, 157.0 mmol),
and 25% aqueous ammonia (18 mL, 128.4 mmol). After stirring for 48
h, the reaction mixture was diluted with 200 mL ethyl acetate and
washed with water, 1N HCl, saturated NaHCO.sub.3, saturated
NH.sub.4 Cl, and brine. Drying over Na.sub.2 SO.sub.4 and
concentration yielded the amide as a tan solid (10.5 g, 64%).
(e) 5-Acetyl-3-(3-chloro-propylsulfanyl)-2-hydroxy-benzamide
Solid aluminum chloride (9.0g, 67.7 mmol) was suspended in 20 mL of
dry dichloromethane at 0.degree. C. The
7,8-dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carboxylic acid
amide (2.7 g, 12.7 mmol) was added as a solution in 20 mL
dichloromethane. The deep green solution was stirred at 0.degree.
C. for 10 min, then neat acetyl chloride (10 mL, 140.6 mmol) was
added dropwise with stirring. The suspension was stirred at
0.degree. C. for 20 min, then at rt for 30 h. The reaction was
quenched with 4 N HCl and extracted repeatedly with ethyl acetate.
Drying over Na.sub.2 SO.sub.4 and concentration yielded the crude
product. Sgc (ethyl acetate) yielded the product as a tan solid
(1.6 g, 44%). MS [M-H].sup.- 286.
(f)
2-Acetyl-7,8-dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carboxylic
Acid Amide
The 5-acetyl-3-(3-chloro-propylsulfanyl)-2-hydroxy-benzamide (2.87
g, 10 mmol) was dissolved in 8 mL dry DMF. Solid Cs.sub.2 CO.sub.3
(4.93 g, 15.1 mmol) was added, followed by catalytic amounts of KI
(0.1 g). The suspension was warmed to 70.degree. C. under nitrogen
and was stirred for 72 h. After cooling, it was diluted with ethyl
acetate and enough 4 N HCl to make the pH about 2. The aqueous
layer was extracted with more ethyl acetate. The combined organic
layers were washed with water and brine. Drying over Na.sub.2
SO.sub.4 and concentration yielded the product as a solid (1 g,
40%).
(g)
2-(1-Hydroxy-ethyl)-7,8-dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carbox
ylic Acid Amide
To
2-acetyl-7,8-dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carboxylic
acid amide (0.40 g, 1.59 mmol) suspended in EtOH (10 mL) was added
NaBH.sub.4 (0.060 g, 1.59 mmol). The mixture was stirred for 5 min,
made acidic with 1 N HCl, and the EtOH removed in vacuuo. The
aqueous was extracted with EtOAc. The combined extracts were washed
with water, dried over magnesium sulfate and concentrated to a foam
(0.35 g, 87%). MS [M+H].sup.+ 252.
(h) 2-(1-Azido-ethyl)-7,8-dihydro-6
H-5-oxa-9-thia-benzocycloheptene-4-carboxylic Acid Amide
This compound was prepared as for example 1(d). (0.26 g, 66%).
(i)
2-(1-Amino-ethyl)-7,8-dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carboxyl
ic Acid Amide
To
2-(1-azido-ethyl)-7,8-dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carboxyl
ic acid amide (0.20 g, 0.73 mmol) in THF (5 mL) was added water
(0.10 mL) and triphenylphosphine (0.19 g, 0.73 mmol). The mixture
was heated to 50.degree. C. for 20 h, evaporated, and
chomatographed over silica gel (10% MeOH/CHCl.sub.3) to give a
colorless oil (0.10 g, 55%).
(j)
[(4-{(S)-2-Acetylamino-(S)-2-[1-(4-carbamoyl-7,8-dihydro-6H-5-oxa-9-thia-b
enzocycylohepten-2-yl)-ethylcarbamoyl]-ethyl}-phenyl)-difluoro-methyl]-phos
phonic Acid
This compound was prepared as for example 3 (a-b). MS [M+H].sup.+
572.
Example 20
{4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro
-5H-benzocyclohepten-(5-5-ylcarbamoyl)-ethyl]-2-phosphono-phenoxy}-acetic
acid and
[4-[(S-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro-5
H-benzocyclohepten-(5-5-ylcarbamoyl)-ethyl]-2-(ethoxy-hydroxy-phosphoryl)-p
henoxy]-acetic acid ##STR39##
(a) Phosphoric Acid Diethyl Ester 2-iodo-phenyl Ester
To a mixture of 2-iodophenol (14.1 g, 64.1 mmol) and potassium
carbonate (17.6 g, 128 mmol) in MeCN (100 mL) was added
diethylchlorophosphate (11.1 mL, 76.7 mmol). The mixture was
allowed to stir at rt for 5 h. The solvent was removed under
reduced pressure, the residue diluted with water and extracted with
EtOAc. The organic layer was washed with brine, dried over
anhydrous Na.sub.2 SO.sub.4 and concentrated under reduced
pressure. The residue was chromatographed over silica gel (elution
with a stepwise gradient 25-40% EtOAc-hexanes) to give 21.7 g (95%)
of a pale oil. R.sub.f 0.47 (1:1 EtOAc-hexanes). Electrospray Mass
Spectrum (50/50 acetonitrile/water) m/z 357 (M+H).
(b) (2-hydroxy-phenyl)-phosphonic Acid Diethyl Ester
Casteel, D. A.; Peri, S. P. Synthesis, 1991, 691.
To a cooled (-78.degree. C.) solution of phosphoric acid diethyl
ester 2-iodo-phenyl ester (21.7 g, 61.0 mmol) in dry THF (500 mL)
under N.sub.2 was added a 2.5 M solution of BuLi (40 mL, 100 mmol).
After 20 min, the reaction was treated with satd aq NH.sub.4 Cl (50
mL) and allowed to warm to rt. The mixture was diluted with H.sub.2
O (50 mL) and extracted twice with EtOAc. The organic extracts were
pooled, washed with brine, dried over anhydrous Na.sub.2 SO.sub.4
and concentrated under reduced pressure. The residue was
chromatographed over silica gel (20% EtOAc-hexanes) to give 13.1 g
(93%) of a pale oil. R.sub.f 0.60 (1:1 EtOAc-hexanes). Electrospray
Mass Spectrum (50/50 acetonitrile/water) m/z 231 (M+H).
(c) (2-hydroxy-5-iodo-phenyl)-phosphonic Acid Diethyl Ester
To a cooled (0.degree. C.) mixture of (2-hydroxy-phenyl)-phosphonic
acid diethyl ester (13.1 g, 56.9 mmol) and sodium iodide (10.2 g,
68.3 mmol) in DMF (200 mL) was added chloramine-T trihydrate (19.2
g, 68.3 mmol) over 5 min. After 10 min, the reaction was allowed to
warm to rt and the mixture was allowed to stir at rt for 2 h. The
mixture was diluted with H.sub.2 O (50 mL), acidified using 0.5 N
HCl and extracted twice with EtOAc. The organic extracts were
pooled and washed sequentially with satd. Na.sub.2 S.sub.2 O.sub.3
/brine (1/1) and then brine. The organic layer was dried over
anhydrous Na.sub.2 SO.sub.4 and concentrated under reduced
pressure. The residue was chromatographed over silica gel (elution
with a stepwise gradient 5-15% EtOAc-hexanes) to give 14.9 g (73%)
of a colorless solid after recrystallization (EtOAc-hexanes).
R.sub.f 0.30 (20% EtOAc-hexanes). Electrospray Mass Spectrum (50/50
acetonitrile/water) m/z 357 (M+H).
(d) (2-benzyloxy-5-iodo-phenyl)-phosphonic Acid Diethyl Ester
To a mixture of (2-hydroxy-5-iodo-phenyl)-phosphonic acid diethyl
ester (14.7 g, 41.2 mmol) and cesium carbonate, (17.4 g, 53.5 mmol)
in DMF (150 mL) was added benzyl bromide (6.40 mL, 54 mmol). The
reaction was allowed to stir for 2.5 days at rt at which point the
reaction was concentrated under reduced pressure, the residue
diluted in 0.5 N HCl (30 mL) and extracted twice with EtOAc. The
pooled organic extracts were washed with brine, dried over
anhydrous Na.sub.2 SO.sub.4 and concentrated under reduced pressure
to give 17.4 g (91%) of a colorless solid after recrystallization
(EtOAc-hexanes). R.sub.f 0.36 (1:1 EtOAc-hexanes). Electrospray
Mass Spectrum (50/50 acetonitrile/water) m/z 447 (M+H).
(e)3-[4-benzyloxy-3-(diethoxy-phosphoryl)-phenyl]-(S)-2-tert-butoxycarbonyl
amino-propionic Acid Methyl Ester
To a mixture of Zn dust (403 mg, 6.17 mmol) in dry DMA (1 mL) and
dry THF (1 mL) was added dibromoethane (55 microliters, 0.64 mmol)
and chlorotrimethylsilane (80 microliters, 0.63 mmol). The mixture
was sonicated for 15 min under N.sub.2. To this mixture was added a
solution of Boc-L-iodoalanine-methyl ester (1.45 g, 4.41 mmol) in
dry DMA (1 mL) and dry THF (1 mL). The mixture was sonicated for 30
min and then heated (65.degree. C.) for 45 min under N.sub.2. To
the heated mixture was added a solution
(2-benzyloxy-5-iodo-phenyl)-phosphonic acid diethyl ester (1.37 g,
2.94 mmol), bis(benzonitrile)dichloropalladium (II) (67 mg, 0.17
mmol) and tri-ortho-tolylphosphine (148 mg, 0.486 mmol) in dry
DMA(1 mL) and THF (1 mL) over 5 min. The mixture was allowed to
stir for 3 h at 65.degree. C. under N.sub.2 at which point the
reaction was allowed to cool to rt, diluted with 0.5 N HCl and
extracted twice with Et.sub.2 O. The organic extracts were pooled,
washed with brine, filtered, dried over anhydrous MgSO.sub.4 and
concentrated under reduced pressure. An initial chromatographic
purification over silica gel (elution with a stepwise gradient
15-40% acetone-hexanes) was followed by a second chromatographic
purification over silica gel (70% EtOAc-hexanes) to give 958 mg
(63%) of a pale oil R.sub.f 0.33 (80% EtOAc-hexanes). Electrospray
Mass Spectrum (50/50 acetonitrile/water) m/z 522.5 (M+H).
(f)
(s)-2-tert-butoxycarbonylamino-3-[4-benzyloxy-3-(diethoxy-phosphoryl)-phen
yl]-propionic Acid
To a cooled (0.degree. C.) solution
(S)-2-tert-butoxycarbonylamino-3-[3-(diethoxy-phosphoryl)-4-hydroxy-phenyl
]-propionic acid methyl ester (423 mg, 0.98 mmol) in MeOH (4 mL)
was added lithium hydroxide monohydrate (84 mg, 2.0 mmol) in
H.sub.2 O (4 mL). After 1 h, the reaction was allowed to warm to
rt. After 2.5 h, the solution was carefully acidified to pH 2 using
0.5 N HCl and extracted twice with EtOAc. The organic extracts were
pooled, washed with brine, dried over anhydrous Na.sub.2 SO.sub.4
and concentrated to give 379 mg (91%) of a yellow foam. The crude
material was carried on without further purification. Electrospray
Mass Spectrum (50/50 acetonitrile/water) m/z 416 (M-H).
(g)(S)-9-amino-3-cylohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2
-carboxylic Acid Amide
To a cooled (0.degree. C.) solution of
(S)-9-tert-butoxycarbonylamino-3-cycycloxylmethoxy-6,7,8,9-tetrahydro--SH-
benzocycloheptene-2-carboxylic acid amide (198 mg, 0.475 mmol) in
DCM (1 mL) was added TFA (1 mL). After 10 min, the reaction was
allowed to warm to rt and after 1 h, the reaction was evaporated to
dryness. The material was used without further purification for the
next step.
(h)
{2-Benzyloxy-5-[(5-2-tert-butoxycarbonylamino-2-(3-carbamoyl-2-cycycloxylm
ethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(!2-5-ylcarbamoyl)-ethyl]-phe
nyl}-phosphonic Acid Diethyl Ester
A solution of
(S)-2-tert-butoxycarbonylamino-3-[4-benzyloxy-3-(diethoxy-phosphoryl)-phen
yl]-propionic acid (355 mg, 0.700 mmol),
1-hydroxy-7-azabenzotriazole (123 mg, 0.904 mmol) and
(S)-9-amino-3-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-
carboxylic acid amide (ca. 0.475 mmol) in DMF (5 mL) was
neutralized by dropwise addition of 4-methylmorpholine. The
solution was cooled (-20.degree. C.) and EDC (173 mg, 903 mmol) was
added. The reaction was allowed to slowly warm to rt under N.sub.2.
After 16 h, the reaction was diluted with 0.5 N HCl and extracted
twice with EtOAc. The pooled organic extracts were washed
sequentially with brine, satd. NaHCO.sub.3 and brine. The organic
layer was dried over Na.sub.2 SO.sub.4 and concentrated. The
residue was chromatographed over silica gel (elution with 2%
MeOH/CHCl.sub.3) to give 342 mg (89%) of a colorless solid. R.sub.f
0.59 (EtOAc). Electrospray Mass Spectrum (50/50 acetonitrile/water)
m/z 806 (M+H).
(i){5-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahy
dro-5H-benzocyclohepten-(S)-5-ylcarbamoyal-ethyl]-2-benzyloxy-phenyl}-phosp
honic Acid Diethyl Ester
To a cooled (0.degree. C.) solution of
{2-benzyloxy-5-[(S)-2-tert-butoxycarbonylamino-2-(3-carbamoyl-2-cycycloxyl
methoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-ph
enyl}-phosphonic acid diethyl ester (342 mg, 0.425 mmol) in DCM (1
mL) was added TFA (1 mL). After 10 min, the reaction was allowed to
warm to rt and after 1 h, the reaction was evaporated to dryness.
The crude material was dissolved in DCM (3 m) and
4-methylmorpholine (70 .mu.L, 0.638 mmol) and acetic anhydride (60
.mu.L, 0.638 mmol) were added. The reaction was allowed to stir at
rt for 17 h. The reaction was diluted with 0.5N HCl and extracted
twice with EtOAc. The combined extracts were washed with brine,
dried over Na.sub.2 SO.sub.4 and concentrated to a colorless oil.
The material was used without further purification for the next
step.
(j)[4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahy
dro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-(diethoxy-phosphoryl)-p
henoxy]-acetic Acid tert-butyl Ester
To a solution of
{5-[(S)-2-acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydr
o-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-benzyloxy-phenyl}-phospho
nic acid diethyl ester (ca. 0.425 mmol) in MeOH (10 mL) was added
10% Pd/C (4 mg). The heterogeneous mixture was degassed under
reduced pressure and allowed to stir at rt for 18 h under H.sub.2.
The catalyst was removed by filtration and the filtrate
concentrated. The residue was dissolved in MeCN (5 mL) and cesium
carbonate(0.64 mmoles) and tert-butylbromoacetate (0.64 mmoles) was
added. The mixture was allowed to stir at rt for 17 h. The reaction
was diluted with water and extracted twice with EtOAc. The combined
extracts were washed with brine, dried over Na.sub.2 SO.sub.4 and
concentrated. The residue was chromatographed over silica gel
(elution with 2% MeOH/CHCl.sub.3) to give 278 mg (85%) of a
colorless solid. R.sub.f 0.21 (5% MeOH/CHCl.sub.3). Electrospray
Mass Spectrum (50/50 acetonitrile/water) m/z 770 (M-H).
(k)
{4-[(S-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro
-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-phosphono-phenoxy}-acetic
Acid and
[4-[(S)-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro-
5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-(ethoxy-hydroxy-phosphoryl)
-phenoxy-acetic Acid
To a cooled (0.degree. C.) solution of
[4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydr
o-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-(diethoxy-phosphoryl)-phe
noxy]-acetic acid tert-butyl ester (200 mg, 0.259 mmol) in DCM (1
mL) was added TFA (1 mL). After 10 min, the reaction was allowed to
warm to rt and after 1 h, the reaction was evaporated to dryness.
The residue was dissolved in MeCN (3 mL) and cooled (0.degree. C.).
To this solution was added dropwise iodotrimethylsilane (0.80 mL,
5.9 mmol). After 5 h at 0.degree. C., the reaction was quenched
with 20% NaHSO.sub.3. A solution of 10% NaOH was added until the
mixture appeared colorless. The mixture was filtered and purified
by RP-HPLC (CH.sub.3 CN/H.sub.2 O) to give
{4-[(5)-2-acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydr
o-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-phosphono-phenoxy]-acetic
acid (45 mg) MS 658 (M-H) and
[4-[(S)-acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro-
5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-(ethoxy-hydroxy-phosphoryl)
-phenoxy]-acetic acid (25 mg) MS 686 (M-H).
Example 21
{4-[(S-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro-
5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-carbomethoxy-phenoxyl-acetic
Acid ##STR40##
(a)
[1-(3-Carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten
-(S-5-ylcarbamoyl)-(5-2-(3,4-dihydroxy-phenyl)-ethyl]-carbamic acid
tert-butyl ester
(S)-2-tert-Butoxycarbonylamino-3-(3,4-dihydroxy-phenyl)-propionic
acid dicycycloxylamine (125 mg, 0.300 mmol) was coupled to
(S)-9-amino-3-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-
carboxylic acid amide as described for Example X (h). The product
was obtained as a colorless foam (60 mg, 33%). R.sub.f 0.32 (5%
MeOH/CHCl.sub.3). Electrospray Mass Spectrum (50/50
acetonitrile/water) m/z 594 (M-H).
(b
14-[(5)-2-tert-Butoxycarbonylamino-2-(3-carbamoyl-2-cylcohexylmethoxy-6,7,
8,9-tetrahydro-5H-benzocyclohepten-(S-5-ylcarbamoyl)-ethyl]-2-tert-butoxyca
rbonylmethoxy-phenoxy}-acetic Acid tert-butyl Ester
To a solution of
[1-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten
-(S)-5-ylcarbamoyl)-(S)-2-(3,4-dihydroxy-phenyl)-ethyl]-carbamic
acid tert-butyl ester (58 mg, 0.097 mmol) in DMF (1.5 mL) was added
tert-butylbromoacetate (50 .mu.L, 0.34 mmol) and cesium carbonate
(150 mg 0.46 mmol). The mixture was allowed to stir at rt for 22 h.
A second addition of tert-butylbromoacetate (25 .mu.L, 0.17 mmol)
and cesium carbonate (75 mg 0.23 mmol) was necessary to drive the
reaction to completion after an additional 40 h. The mixture was
diluted with EtOAc and 0.5 N HCl. The aqueous portion was extracted
with fresh EtOAc and the combined organic layers were washed with
brine. The solution was dried over Na.sub.2 SO.sub.4, concentrated
and chromatographed over silica gel (elution with 3%
MeOH/CHCl.sub.3) to give the product as a colorless solid after
trituration with EtOAc and hexanes (67 mg, 84%). R.sub.f 0.11 (3%
MeOH/CHCl.sub.3). Electrospray Mass Spectrum (50/50
acetonitrile/water) m/z 824 (M+H).
(c)
(4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydr
o-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-carboxymethoxy-phenoxy}-a
cetic Acid
To a cooled (0.degree. C.) solution of
{4-[(S)-2-tert-butoxycarbonylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,
8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-tert-butoxyc
arbonylmethoxy-phenoxy}-acetic acid tert -butyl ester (66 mg, 0.050
mmol) in DCM (1 mL) was added TFA (1 mL). After 0.5 h, the reaction
was allowed to warm to rt and after 3 h, the reaction was
evaporated to dryness. The crude material was dissolved in DCM (1
mL) and the solution neutralized with 4-methylmorpholine. Acetic
anhydride (10 .mu.L, 0.11 mmol) was added and the reaction allowed
to stir at rt for 18 h. The reaction was concentrated, diluted with
MeCN/H.sub.2 O and purified by RP-HPLC (MeCN/H.sub.2 O) to give the
product as a colorless solid after lyophilization (22 mg, 44%).
Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 654
(M+H).
Example 22
(4-[(s)-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro
-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-carboxymethyl-phenoxy
-acetic Acid ##STR41##
(a)(S-1-tert-Butoxycarbonylamino-3-(4-hydroxy-3-iodo-phenyl)-propionic
Acid
To a solution of L-4-iodotyrosine (2.00 g, 6.51 mmol) in dioxane
(50 mL) and H.sub.2 O (40 mL) was added 4-methylmorpholine (0.70
mL, 6.4 mmol) and di-tert-butyldicarbonate (2.13 g, 9.77 mmol).
After 18 h at rt, di-tert-butyldicarbonate (2.13 g, 9.77 mmol) was
added to drive the reaction to completion. The reaction was allowed
to stir for another 24 h. The solution was acidified with 0.5 N HCl
and extracted twice with EtOAc. The combined extracts were washed
with brine, dried over Na.sub.2 SO.sub.4 and concentrated give the
product as a foamy solid. The material was used without further
purification for the next step.
(b)(S-2-tert-Butoxycarbonylamino-3-(4-hydroxy-3-iodo-phenyl)-propionic
Acid Benzyl Ester
To a solution of
(S)-2-tert-butoxycarbonylamino-3-(4-hydroxy-3-iodo-phenyl)-propionic
acid (3.27 g, 8.03 mmol) in DCM (20 mL) was added DMAP (1.03 g,
8.43 mmol) and benzyl alcohol (2.4 mL, 23 mmol). The solution was
cooled (-20.degree. C.) and EDC (1.62 g, 8.45 mmol) was added. The
reaction was allowed to slowly warm to rt. After 17 h, the reaction
was diluted with 0.5 N HCl and extracted twice with EtOAc. The
combined extracts were washed with brine, dried over Na.sub.2
SO.sub.4 and concentrated. The residue was chromatographed over
silica gel (elution with 15% EtOAc/hexanes) to give the product as
a colorless solid (1.62 g, 50%). R.sub.f 0.41 (40% EtOAc/hexanes).
Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 496
(M-H).
(c)
(S)-2-tert-Butoxycarbonylamino-3-(4-ethoxcycarbonylmethoxy-3-iodo-phenyl-p
ropionic Acid Benzyl Ester
To a solution of (S)-2-tert
butoxycarbonylamino-3-(4-hydroxy-3-iodo-phenyl)-propionic acid
benzyl ester (1.88 g, 3.78 mmol) in MeCN (20 mL) was added cesium
carbonate (1.85 g, 5.68 mmol) and ethylbromoacetate (0.63 mL, 5.7
mmol). The reaction was allowed to stir at rt for 64 h. The
precipitate was removed by filtration and the filtrate
concentrated. The residue was chromatographed over silica gel
(elution with a gradient 15-33% EtOAc/hexanes) to give the product
as a colorless solid (2.00 g, 91%). R.sub.f 0.26 (40%
EtOAc/hexanes). Electrospray Mass Spectrum (50/50
acetonitrile/water) m/z 606 (M+Na).
(d)3-(3-Allyl-4-ethoxycarbonylmethoxy-phenyl)-(M-2-tert-butoxycarbonylamino
-propionic Acid Benzyl Ester
To a solution of
(S)-2-tert-butoxycarbonylamino-3-(4-ethoxycarbonylmethoxy-3-iodo-phenyl)-p
ropionic acid benzyl ester (1.99 g, 3.41 mmol) in DMF (15 mL) was
added lithium chloride (289 mg, 6.82 mmol), all yl tributyltin (1.3
mL, 4.2 mmol) and bis(triphenylphosphine)palladium(II) chloride
(143 mg, 0.204 mmol). The mixture was heated (90.degree. C.) and
allowed to stir under N.sub.2. The reaction was allowed to cool to
rt and diluted with Et.sub.2 O and 0.5 N HCl. The aqueous phase was
extracted with fresh Et.sub.2 O. The combined organic extracts were
stirred with saturated potassium fluoride for 17 h. The organic
layer was dried over MgSO.sub.4, concentrated and chromatographed
(elution with 30% Et.sub.2 O/hexanes). The product was obtained as
a colorless solid after recrytallization (EtOAc/hexanes) (1.14 g,
67%). R.sub.f 0.27 (40% Et.sub.2 O/hexanes). Electrospray Mass
Spectrum (50/50 acetonitrile/water) m/z 520 (M+Na).
(e)(S)-2-tert-Butoxycarbonylamino-3-[4-ethoxycarbonylmethoxy-3-(2-oxo-ethyl
)-phenyl]-propionic Acid Benzyl Ester
To a solution of
3-(3-allyl-4-ethoxycarbonylmethoxy-phenyl)-(S)-2-tert-butoxycarbonylamino-
propionic acid benzyl ester (1.00 g, 2.01 mmol) in THF (20 mL) was
added pyridine (2 drops) and H.sub.2 O (20 mL) followed by osmium
tetroxide (1.2 mL, 2.5% wt. in tBuOH, 0.096 mmol). The mixture was
allowed to stir for 10 min and NaIO.sub.4 was added portionwise
over 0.5 h. The mixture was allowed to stir for another 0.5 h at
rt. The reaction was diluted with H.sub.2 O and extracted twice
with Et.sub.2 O. The combined organic extracts were washed with
brine, dried over MgSO.sub.4 and concentrated. The residue was
chromatographed over silica gel (elution with 25% EtOAc/hexane) to
give the product as an oil (902 mg, 90%). R.sub.f 0.18 (25%
EtOAc/hexanes). Electrospray Mass Spectrum (50/50
acetonitrile/water) m/z 522 (M+Na).
(f)(5-2-tert-Butoxycarbonylamino-3-(4-ethoxycarbonylmethoxy-3-ethoxycarbony
lmethyl-phenyl)-propionic Acid Benzyl Ester
To a solution of
(S)-2-tert-butoxycarbonylamino-3-[4-ethoxycarbonylmethoxy-3-(2-oxo-ethyl)-
phenyl]-propionic acid benzyl ester (880 mg, 1.76 mmol) in tBuOH
(35 mL) and cycycloxane (9 mL) was added over 15 min a solution of
sodium chlorite (1.79 g, 15.8 mmol, 80% tech.) and sodium
dihydrogen phosphate dihydrate (1.92 g, 12.3 mmol) in H.sub.2 O (15
mL). The mixture was allowed to stir at rt for 4 h. The volatiles
were removed in vacuo and the remaining mixture was acidified with
0.5 N HCl and extracted twice with EtOAc. The combined extracts
were washed with brine, dried over Na.sub.2 SO.sub.4 and
concentrated. The residue was dissolved in DMF (5 mL) and cesium
carbonate (2.87 g, 8.80 mmol) and iodoethane (0.70 mL, 8.7 mmol)
were added. The reaction was allowed to stir at rt for 20 h. The
reaction was diluted with EtOAc and 0.5 N HCl. The aqueous layer
was extracted with fresh EtOAc and the combined organic extracts
were washed with brine and dried over Na.sub.2 SO.sub.4. The
solution was concentrated and chromatographed over silica gel
(elution with 25% EtOAc/hexanes) to give the product as a colorless
oil (849 mg, 89%). R.sub.f 0.13 (25% EtOAc/hexanes). Electrospray
Mass Spectrum (50/50 acetonitrile/water) m/z 567 (M+Na).
(g)
(S)-2-tert-Butoxycarbonylamino-3-(4-ethoxycarbonylmethoxy-3-ethoxycarbonyl
methyl-phenyl)-propionic Acid
To a solution of
(S)-2-tert-butoxycarbonylamino-3-(4-ethoxycarbonylmethoxy-3-ethoxycarbonyl
methyl-phenyl)-propionic acid benzyl ester (796 mg, 1.46 mmol) in
MeOH (8 mL) was added 10% Pd/C (4 mg). The heterogeneous mixture
was degassed under reduced pressure and allowed to stir at rt for
18 h under H.sub.2. The catalyst was removed by filtration and the
filtrate evaporated to dryness to give the product as a colorless
foam (515 mg, 78%) Electrospray Mass Spectrum (50/50
acetonitrile/water) m/z 476 (M+Na).
(h)
{4-[(S)-2-tert-Butoxycarbonylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,
8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl-2-ethoxycarbony
lmethyl-phenoxy}-acetic Acid Ethyl Ester
(S)-2-tert-Butoxycarbonylamino-3-(4-ethoxycarbonylmethoxy-3-ethoxycarbonylm
ethyl-phenyl)-propionic acid (65 mg, 0.16 mmol) was coupled to
(S)-9-amino-3-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-
carboxylic acid amide as described for Example X (h). The product
was obtained as a colorless solid (92 mg, 78%). R.sub.f 0.27 (75%
EtOAc/hexane). Electrospray Mass Spectrum (50/50
acetonitrile/water) m/z 774 (M+Na)
(i){4-[(S-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahyd
ro-5H-benzocyclohepten-(S-5-ylcarbamoyl)-ethyl]-2-carboxymethyl-phenoxy}-ac
etic Acid
To a cooled (0.degree. C.) solution of
{4-[(S)-2-tert-Butoxycarbonylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,
8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-ethoxycarbon
ylmethyl-phenoxy}-acetic acid ethyl ester (92 mg, 0.12 mmol) in DCM
(0.5 mL) was added TFA (0.5 mL). After 10 min, the reaction was
allowed to warm to rt and after 1 h, the reaction was evaporated to
dryness. The crude material was dissolved in DCM (0.5 mL) and DMA
(0.5 mL) and 4-methylmorpholine (16 mL, 0.17 mmol) and acetic
anhydride (25 .mu.L, 0.23 mmol) were added. The reaction was
allowed to stir at rt for 18 h. The reaction was diluted with 0.5N
HCl and extracted twice with EtOAc. The combined extracts were
washed with brine, dried over Na.sub.2 SO.sub.4 and concentrated to
a colorless oil. The residue was dissolved in MeOH (0.7 mL) and DMF
(0.7 mL) and cooled (0.degree. C.). To this solution was added
lithium hydroxide monohydrate (31 mg, 0.74 mmol) in H.sub.2 O (0.6
mL). After 0.5 h, the reaction was allowed to warm to rt and after
1.5 h, the reaction was neutralized by careful addition of 6 N HCl.
Purification by RP-HPLC gave the product as a colorless solid after
lyophilization (23 mg, 30%). Electrospray Mass Spectrum (50/50
acetonitrile/water) m/z 638 (M+H)
Example 23
({4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydr
o-5H-benzocyclohepten-(S)-5-ylcarbamoyl-ethyl]-phenyl}-hydroxyacetyl-amino)
-acetic Acid ##STR42##
(a){4-[(S)-2-tert-butoxycarbonylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,
7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-carb
amic Acid Benzyl Ester
3-(4-Benzyloxycarbonylamino-phenyl)-(S)-2-tert-butoxycarbonylamino-propioni
c acid (417 mg, 0.16 mmol) was coupled to
(S)-9-amino-3-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-
carboxylic acid amide as described for Example X (h). The product
was obtained as a colorless solid (231 mg, 65%). R.sub.f 0.28 (5%
MeOH/CHCl.sub.3). Electrospray Mass Spectrum (50/50
acetonitrile/water) m/z 713 (M+H).
(b))({4-[(S)-2-Amino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro-
5H-benzocyclohepten-(S)-5-ylcarbamoyl)thyls-phenyl}-ethoxyacetyl-amino)-ace
tic Acid Ethyl Ester
To a solution of
{4-[(S)-2-tert-butoxycarbonylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,
8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-carbam
ic acid benzyl ester (231 mg, 0.324 mmol) in MeOH (5 mL) was added
10% Pd/C (10 mg). The heterogeneous mixture was degassed under
reduced pressure and allowed to stir at rt for 46 h under H.sub.2.
The catalyst was removed by filtration and the filtrate evaporated
to dryness. The residue was dissolved in MeCN (5 mL) and cesium
carbonate (264 mg, 0.810 mmol) and ethylbromoacetate (0.36 mL, 3.25
mmol) were added. The reaction was warmed (90.degree. C.) and
allowed to stir for 40 h. A second portion of ethylbromoacetate
(0.18 mL was added and the reaction allowed to stir at 90.degree.
C. for 18 h. The reaction was concentrated, diluted with H.sub.2 O
and extracted twice with EtOAc. The combined organic extracts were
washed with brine, dried over Na.sub.2 SO.sub.4 and concentrated.
The residue was chromatographed over silica gel (elution with a
stepwise gradient 2-4% MeOH/CHCl.sub.3) to give the product as a
colorless solid (132 mg, 63%). R.sub.f 0.18 (5% MeOH/CHCl.sub.3).
Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 651
(M+H).
(c)({4-F(6)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrah
ydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-ethoxyacetyl-ami
no)-acetic Acid Ethyl Ester
To a solution of
({4-[(S)-2-amino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydro-5H-
benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-ethoxyacetyl-amino)-acet
ic acid ethyl ester (132 mg, 0.203 mmol) in DCM (1.5 mL) was added
4-methylmorpholine (27 .mu.L, 0.24 mmol) and acetic anhydride (0.23
.mu.L, 0.24 mmol). Allowed to stir at rt for 18 h. The reaction was
diluted with 0.5 N HCl and extracted twice with EtOAc. The combined
extracts were washed with brine, dried over Na.sub.2 SO.sub.4 and
concentrated. The residue was chromatographed over silica gel
(elution with a stepwise gradient 1-3% MeOH/CHCl.sub.3) to give the
product as a colorless solid (95 mg, 68%). R.sub.f 0.41 (5%
MeOH/CHCl.sub.3). Electrospray Mass Spectrum (50/50
acetonitrile/water) m/z 693 (M+H).
(d)({4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrah
ydro-5H-benzocyclohepten-(S-5-ylcarbanol)-ethyl]-phenyl}-hydroxyacetyl-amin
o)-acetic Acid
To a solution of
({4-[(S)-2-acetylamino-2-3-carbamoyl-2-cycycloxylmethoxy-6,7,8,9-tetrahydr
o-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-ethoxyacetyl-amino)
-acetic acid ethyl ester (80 mg, 0.12 mmol) in MeOH (1 mL) was
added lithium hydroxide monohydrate (15 mg, 0.36 mmol) in H.sub.2 O
(1 mL). After 3 h, the reaction was diluted with MeOH (4 mL) and
DMF (1 mL) and purified by RP-HPLC (MeCN/H.sub.2 O) to give the
product as a colorless solid after lyophilization (25 mg, 34%).
Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 635
(M-H).
* * * * *